Patent application title: Compositions useful for and methods of modulating angiogenesis
Inventors:
Richard Lang (Loveland, OH, US)
IPC8 Class: AA61K3817FI
USPC Class:
514 12
Class name: Designated organic active ingredient containing (doai) peptide containing (e.g., protein, peptones, fibrinogen, etc.) doai 25 or more peptide repeating units in known peptide chain structure
Publication date: 2009-02-05
Patent application number: 20090036373
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Patent application title: Compositions useful for and methods of modulating angiogenesis
Inventors:
Richard Lang
Agents:
TAFT, STETTINIUS & HOLLISTER LLP
Assignees:
Origin: CINCINNATI, OH US
IPC8 Class: AA61K3817FI
USPC Class:
514 12
Abstract:
Compositions and methods for treating subjects with disorders
characterized by aberrant vascular endothelial cell growth are provided.
The compositions comprise agents that are combinations of a Wnt pathway
stimulator component and a Tie2 pathway repressor component. Particularly
useful Wnt pathway stimulators include, but are not limited to,
Wnt7b-like molecules. Particularly useful Tie2 pathway repressor
components include, but are not limited to, Ang2-like molecules. The
methods allow for modulation of vascular endothelial cells, vascular
endothelial cell vessels, capillary bed development, and angiogenesis.Claims:
1. A vascular endothelial cell vessel modulating compound comprising a Wnt
pathway stimulator and a Tie2 pathway repressor.
2. The compound of claim 1, wherein the Wnt pathway stimulator is a Wnt7b-like molecule.
3. The compound of claim 2, wherein said Wnt7b-like molecule is a Wnt7b molecule selected from the group consisting of:(a) an isolated polypeptide having an amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:3, SEQ ID:5, or SEQ ID NO:6; and(b) an isolated polypeptide having an amino acid sequence having at least 95% identity to an amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:3, SEQ ID:5, or SEQ ID NO:6, wherein said polypeptide is capable of interacting with a Fzd4 receptor.
4. The compound of claim 1, wherein the Tie2 pathway repressor is an Ang2-like molecule.
5. The compound of claim 4, wherein said Ang2-like molecule is an Ang2 molecule selected from the group consisting of:(a) an isolated polypeptide having an amino acid sequence set forth in SEQ ID NO:8, SEQ ID NO:9, SEQ ID:11, or SEQ ID NO:12; and(b) an isolated polypeptide having an amino acid sequence having at least 95% identity to an amino acid sequence set forth in SEQ ID NO:8, SEQ ID NO:9, SEQ ID:11, or SEQ ID NO:12, wherein said polypeptide is capable of interacting with Tie2.
6. The compound of claim 1, wherein the Wnt pathway stimulator is a Wnt7b-like molecule and wherein the Tie2 pathway repressor is an Ang2-like molecule.
7. A method of modulating angiogenesis comprising the step of administering a compound of claim 1.
8. A method of modulating a vascular endothelial cell vessel comprising the step of administering a compound of claim 1.
9. A method of modulating a vascular endothelial cell vessel comprising the step of administering a compound of claim 1, wherein said vascular endothelial cell vessel regresses.
10. A vascular endothelial cell vessel modulating compound comprising a Wnt pathway stimulator and a Tie2 pathway repressor, wherein said Wnt pathway stimulator is a Wnt7b-like molecule and wherein the Tie2 pathway repressor is an Ang2-like molecule.
11. A method of modulating angiogenesis comprising the step of administering a compound of claim 10.
12. The method of claim 11 further comprising the step of evaluating the modulation of a vascular endothelial cell vessel subsequent to the administration of said compound.
13. A method of modulating apoptosis of a vascular endothelial cell comprising the step of administering a compound of claim 10, wherein said vascular endothelial cell undergoes apoptosis.
14. A method of modulating a vascular endothelial cell vessel comprising the step of administering a vascular endothelial cell vessel modulating compound comprising a Wnt pathway stimulator and a Tie2 pathway repressor, wherein said Wnt pathway stimulator is a Wnt7b-like molecule and wherein the Tie2 pathway repressor is an Ang2-like molecule.
15. The method of claim 14, wherein said vascular endothelial cell vessel regresses.
16. The method of claim 14, wherein a vascular endothelial cell of said vascular endothelial cell vessel undergoes apoptosis.
17. A method of modulating an angiogenic-related disorder comprising the steps of:(a) identifying a subject exhibiting an angiogenic-related disorder; and(b) administering a therapeutically effective amount of a compound comprising a Wnt pathway stimulator and a Tie2 pathway repressor to said subject.
18. The method of claim 17, wherein said angiogenic-related disorder is selected from the group consisting of ocular angiogenic disorders and hyperproliferative disorders.
19. The method of claim 17, wherein said Wnt pathway stimulator is a Wnt7b-like molecule and wherein the Tie2 pathway repressor is an Ang2-like molecule.
20. The method of claim 17, wherein said angiogenic-related disorder is a vascular endothelial cell vessel-related disorder.
21. The method of claim 20, wherein a vascular endothelial cell vessel regresses.
22. The method of claim 20, wherein a vascular endothelial cell undergoes apoptosis.
23. The method of claim 19, wherein said Wnt7b-like molecule is a Wnt7b molecule selected from the group consisting of:(a) an isolated polypeptide having an amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:3, SEQ ID:5, or SEQ ID NO:6; and(b) an isolated polypeptide having an amino acid sequence having at least 95% identity to an amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:3, SEQ ID:5, or SEQ ID NO:6, wherein said polypeptide is capable of interacting with a Fzd4 receptor.
24. The method of claim 19, wherein said Ang2-like molecule is an Ang2 molecule selected from the group consisting of:(a) an isolated polypeptide having an amino acid sequence set forth in SEQ ID NO:8, SEQ ID NO:9, SEQ ID:11, or SEQ ID NO:12; and(b) an isolated polypeptide having an amino acid sequence having at least 95% identity to an amino acid sequence set forth in SEQ ID NO:8, SEQ ID NO:9, SEQ ID:11, or SEQ ID NO:12, wherein said polypeptide is capable of interacting with Tie2.
25. A method of modulating capillary bed development comprising the steps of(a) providing a subject in need of capillary bed development modulation; and(b) administering a vascular endothelial cell vessel modulating compound comprising a Wnt pathway stimulator and a Tie2 pathway repressor to a subject.
26. A method of modulating a vascular endothelial cell vessel-related disorder comprising the steps of:(a) identifying a subject exhibiting a vascular endothelial cell vessel-related disorder; and(b) administering a Wnt7b expression modulating agent to said subject.
Description:
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001]This application is a continuation of and claims priority to and benefit of PCT US06/34926, filed on Sep. 8, 2006, and U.S. Provisional Patent Application No. 60/714,970, filed on Sep. 8, 2005, which are incorporated herein by reference in their entirety.
FIELD OF THE INVENTION
[0002]The invention relates to the field of modulation of vascular endothelial cell vessels and angiogenesis.
BACKGROUND OF THE INVENTION
[0003]Macrophages have a critical role in inflammatory and immune responses through their ability to recognize and engulf apoptotic cells (Savill et al. (2002) Nature Rev. Immunol. 2:965-975, herein incorporated by reference in its entirety). It is widely believed that macrophage involvement in programmed cell death occurs after the apoptotic event in response to membrane-tethered or soluble "eat-me" signals from dead and dying cells. However, in some circumstances phagocytes actively induce programmed cell death. In mice, macrophages are required for the programmed regression of temporary capillary networks within the developing eye (Lang, R. A. & Bishop, M. J. (1993) Cell 74:453-462 and Diez-Roux, G. & Lang, R. A. (1997) Development 124:3633-3638, herein incorporated by reference in their entirety). The mechanisms governing macrophage involvement in programmed cell death have not been clarified.
[0004]The Wingless (Wnt) signaling pathway has a crucial function in developmental cell fate decisions, and when aberrantly activated, in the development of cancer (Bienz, M & Clevers, H. (2000) Cell 103:311-30). In vertebrates, the canonical Wnt response requires a receptor complex comprising the co-receptor Lrp5 or Lrp6 and a multi-transmembrane pass receptor of the frizzled family. When activated by a Wnt ligand, this complex initiates a cascade of events that culminates in stabilization of β-catenin, its association with Lef/Tcf family of transcription factors and the regulation of target genes including some that stimulate cell cycle entry (see Nusse (1999) Trends Genet 15:1-3; Bienz & Clevers (2000) Cell 103:311-320; He et al. (2004) Development 131:1663-1677; Perrimon, N. (1996) Cell 86:513-516; Behrens et al. (1996) Nature 382:638-642, herein incorporated by reference in their entirety).
[0005]The Wnt gene family includes at least ten genes that encode structurally related secreted glycoproteins. Members of the Wnt family are reported to be regulators of mammary cell growth and differentiation. For example, dysregulation of Wnt signaling has been reported to cause developmental defects and to be implicated in the genesis of several human cancers. Overexpression of Wnt-7b proteins can result in cellular transformation of C57MG cells. Higher Wnt7b expression levels have also been reported in superficial bladder cancer cells as compared to invasive bladder cancer cells. These results suggest that the Wnt7b polypeptide is involved in the early events of bladder tumorigenesis (U.S. Pat. No. 6,653,448 B1, herein incorporated by reference in its entirety).
[0006]Angiopoietins are a family of vascular growth factors that collaborate with members of the vascular endothelial growth factor family to regulate vascular and lymphatic vessel growth, acting via the endothelial receptor tyrosine kinase Tie 2. Although angiopoietin-1 seems to be an obligate activator of the Tie2 receptor, angiopoietin-2 (Ang2) seems to have context specific effects, activating this receptor on some cells while blocking Tie2 activation on other cells or under other conditions. Ang2 is a context-dependent agonist/antagonist of Tie2 and displays a similar multimerization state to angiopoietin-1. See Davis et al. (2003) Nature Structural Biology 10:38-44, herein incorporated by reference in its entirety.
[0007]Thus, a mechanistic assessment of Wnt7b and Ang2's in vivo role is desirable. It is of importance to develop methods of modulating vascular endothelial cell vessel formation. It is of further importance to develop methods of modulating Wnt7b expression.
SUMMARY OF THE INVENTION
[0008]Compositions useful for and methods of modulating a vascular endothelial cell vessel are provided. The inventions are based on the novel discovery that activation of the canonical Wingless (Wnt) signaling pathway and repression of the Tie2 signaling pathway induces apoptosis or programmed cell death in vascular endothelial cells. This discovery counters the widely held notion that Wnt7b facilitates the early events of tumorigenesis. Exposure of a vascular endothelial cell to a Wnt pathway stimulator, such as Wnt7b, and a Tie2 pathway repressor, such as Angiopoietin-2 (Ang2), induces programmed cell death.
[0009]Compositions of the invention include a vascular endothelial cell vessel modulating compound comprising a Wnt pathway stimulator and a Tie2 pathway repressor. In an aspect of the invention, the Wnt pathway stimulator is a Wnt7b-like molecule. In an aspect of the invention, the Wnt7b-like molecule is Wnt7b or a Wnt7b fragment or variant thereof. In an aspect of the invention, the Tie2 pathway repressor is an Ang2-like molecule. In an aspect of the invention, the Ang2-like molecule is Ang2 or an Ang2 fragment or variant thereof. In an aspect of the invention, the Wnt pathway stimulator is a Wnt7b-like molecule and the Tie2 pathway repressor is an Ang2-like molecule. When administered, the compositions of the invention find use in modulating angiogenesis, vascular endothelial cell vessels, and apoptosis of a vascular endothelial cell. In aspects of the methods of modulating a vascular endothelial cell vessel, the vascular endothelial cell vessel regresses. In aspects of the methods of modulating apoptosis of a vascular endothelial cell, the vascular endothelial cell undergoes apoptosis.
[0010]An embodiment of the invention provides methods of modulating a vascular endothelial cell vessel comprising the step of administering a vascular endothelial cell vessel modulating compound. In an aspect of the method, the vascular endothelial cell vessel regresses. In an aspect of the method, a vascular endothelial cell of the vascular endothelial cell vessel undergoes apoptosis.
[0011]An embodiment of the invention provides methods of modulating an angiogenic-related disorder. The methods comprise the steps of identifying a subject exhibiting an angiogenic related disorder and administering a therapeutically effective amount of a compound comprising a Wnt pathway stimulator and a Tie2 pathway repressor to the subject. In an aspect of the invention, the angiogenic related disorder is selected from the group consisting of, but not limited to, ocular angiogenic disorders and hyperproliferative disorders. In an aspect of the invention the Wnt pathway stimulator is a Wnt7b-like molecule and the Tie2 pathway repressor is an Ang2-like molecule. In an aspect of the invention the angiogenic related disorder is vascular endothelial cell vessel-related disorder. In an embodiment a vascular endothelial cell vessel regresses. In an aspect of the method, a vascular endothelial cell of the vascular endothelial cell vessel undergoes apoptosis.
[0012]An embodiment of the invention provides methods of modulating capillary bed development. The methods comprise the step of administering a vascular endothelial cell vessel modulating compound comprising a Wnt pathway stimulator and a Tie2 pathway repressor to a subject.
[0013]Another embodiment of the invention provides methods of modulating a vascular endothelial cell vessel-related disorder. The methods comprise the step of administering a Wnt7b expression modulating agent.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014]FIG. 1 presents an assessment of the effect of macrophage cells on hyaloid vessel regression. Details of the experiment are presented elsewhere herein. Panels A-D present micrographs of hyaloid vessel preparations from wild-type (Panels A and C) and PU.1.sup.-/- (Panels B and D) mice at P5. The micrographs in Panels A and B indicate results obtained from differential interference contrast illumination of the preparations. Solid circles indicate macrophages. The micrographs in Panels C and D indicate results obtained from fluorescent immunostaining with F4/80, a macrophage specific marker, and nuclei-specific marker. Panels A-D are at 400× magnification.
[0015]Panels E-H present micrographs of hyaloid vessel preparations stained with Hoechst 33258. The vessels in panels E and F were obtained from wild-type mice at ages P3 and P8. The vessels in panels G and H were obtained from PU.1.sup.-/- mice at ages P3 and P8. Panels E-H are at 50× magnification.
[0016]Panel I presents a graphical summary of hyaloid vessel number (y-axis) in P8 mice. Column 1 indicates the mean number of hyaloid vessels in wild-type mice; column 2 indicates the mean number of hyaloid vessels in PU.1.sup.+/- mice; column 3 indicates the mean number of hyaloid vessels in PU.1.sup.+/- mice. Error bars indicate standard error.
[0017]FIG. 2 presents the results of experiments performed to assess the role of the Wnt pathway response in vascular endothelial cells (VEC) and hyaloid vessel regression. Panels A-F present micrographs of Hoechst 33258 stained hyaloid vessel preparations obtained from the indicated mice at a 50× magnification. The vessels in Panels A and B were obtained from wild-type mice at P3 and P8 respectively. The vessels in Panels C and D were obtained from Lrp5.sup.lacZ/lacZ mice at P3 and P8 respectively. The vessels in Panels E and F were obtained from Lef1.sup.-/- mice at P3 and P8 respectively.
[0018]The graphs in panels G-I summarize the relative vessel number (Panel G), the number of apoptotic events (Panel H), and the relative proportion of 5'-bromo-deoxyuridine (BrdU)-Positive cells (Panel I) in five mice strains at the indicated age. Results from wild-type (WT) mice are indicated with solid bars; results from Lrp5.sup.+/- mice are indicated with hatched bars; results from Lrp5.sup.-/- mice are indicated with counter-hatched bars; results from Lef1.sup.+/- mice are indicated with empty bars; and results from Lef1.sup.-/- mice are indicated with cross-hatched bars. In Panel I, results obtained from primary and secondary hyaloid capillary branches are indicated separately.
[0019]Panels J-O present micrographs of X-gal stained hyaloid vessels from mice of the indicated genotype and age. Panel J is at a 1000× magnification; Panels K-O are at 630× magnification. Intensely stained TOPGAL expressing cells are indicated by arrowheads; resident macrophages are indicated by dashed circles. The vessels in Panels J-M were obtained from TOPGAL mice at P7 (Panels J-K), P3 (Panel L), and P5 (Panel M). The vessels in Panel N were obtained from Lrp5.sup.lacZ/lacZ mice at P7. The vessels in Panel O were obtained from Lrp5.sup.lacZ/lacZ/TOPGAL mice at P7.
[0020]FIG. 3 presents results from experiments investigating Wnt7b expression in macrophages and involvement in hyaloid cell regression. Panels A-C present micrographs (630× magnification) of X-gal stained hyaloid vessels from Wnt7b.sup.+/lacZ mice at P1, P3, and P5 respectively. Dashed circles indicate macrophages. Micrographs of vessels from wild-type (WT) mice at the indicated ages are shown in Panels D, E, and I. Micrographs of vessels from Wnt7b.sup.d1/d1 mice at the indicated ages are shown in Panels F, G, and J. The preparations in Panels D-G were stained with Hoechst 33258. The preparations in Panels I-J were stained with anti-F4/80.
[0021]The graphs in Panels H and K-L summarize quantification in hyaloid vessels of the vessel number at P8 (Panel H), apoptotic events at P5 (Panel K), and the relative proportion of BrdU-positive cells at P5 (Panel L). Results from wild-type (WT) mice are indicated with solid bars; results from Lrp5.sup.-/- mice are indicated with counter-hatched bars; results from Wnt7.sup.bd1/d1 mice are indicated with empty bars. In Panel L, results obtained from primary and secondary hyaloid capillary branches are indicated separately.
[0022]FIG. 4 presents results from experiments investigating Wnt7b expression in macrophages and involvement in hyaloid cell regression. Panels A and B present micrographs of hyaloid vessels stained with X-gal obtained from TOPGAL and TOPGAL/Wnt7b.sup.d1/d1 mice, respectively. Arrowheads indicate stained cells.
[0023]Panel C summarizes the relative number of X-gal stained cells in hyaloid preparations at P8 from TOPGAL (solid bar) and TOPGAL/Wnt7b.sup.d1/d1 (empty bar) mice. The data are normalized for the increased vessel number of the Wnt7b.sup.d1/d1 mice.
[0024]Panel D summarizes the light units produced by SuperTopFlash cells transfected with Fzd4 and Lrp5 in the presence (+) or absence (-) of Wnt7b producer cells. The data in the left two columns were obtained from experiments performed under conditions which restricted mixing of the two cell types (separated). The data in the right two columns were obtained from experiments performed under conditions which allowed mixing of the two cell types (mixed).
[0025]Panels E-G present micrographs of hyaloid vessel preparations stained with F4/80, vascular endothelial cell cadherin, and nuclei. The hyaloid vessel preparations were obtained from PU.1.sup.-/- mice either uninjected (Panel E), injected with wild-type macrophages (WT mac, Panel F), or injected with Wnt7b.sup.d1/d1 derived macrophages (Wnt7b.sup.d1/d1 mac, Panel G). Panel H presents a graphical summary of the vessel number at P8 in uninjected PU.1.sup.-/- (empty bar) mice, PU.1.sup.-/- mice injected with wild-type macrophages (hatched bar), or PU.1.sup.-/- mice injected with Wnt7b.sup.d1/d1 derived macrophages (cross-hatched bar). Error bars indicate standard error.
DETAILED DESCRIPTION OF THE INVENTION
[0026]The invention provides compositions and methods for modulating vascular endothelial cells, vascular endothelial cell vessels, capillary vessel development, capillary bed development, and angiogenesis. Additionally, the invention provides methods of modulating vascular endothelial cell-related and angiogenic-related disorders. The compositions and methods of the invention were developed from investigations that revealed that Wnt7b and angiopoietin-2 (Ang-2) modulate the regression of vascular endothelial vessels. Prior work in the field suggested that Wnt7b facilitates the early stages of tumorigenesis (U.S. Pat. No. 6,653,448, herein incorporated by reference in its entirety). Prior to this work it was recognized that the Wnt signaling pathway is often a stimulus for proliferation; thus the results of the investigations described herein were unexpected.
[0027]Compositions of the invention include a vascular endothelial cell vessel modulating compound. A "vascular endothelial cell vessel modulating compound" of the invention comprises a Wnt pathway stimulator component and a Tie2 pathway repressor component. By "Wnt pathway stimulator" is intended any agonist of the Fzd4 receptor, any Dv1 activator, any GSK3 repressor, any β-catenin modulator, or any Lef1 stimulator that results in increased myc and cyclinD1 expression or results in advancement of the cell through the cell cycle. Wnt pathway stimulators include but are not limited to Wnt7b-like molecules and Wnt7b expression modulating agents.
[0028]By "Wnt7b-like molecule" is intended an agent capable of interacting with a Fzd4 receptor. Wnt7b-like molecules include, but are not limited to, an isolated nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:1; an isolated nucleic acid molecule having a nucleotide sequence that encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:3; an isolated nucleic acid molecule having a nucleotide sequence that encodes a polypeptide having at least 95% identity to the amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:3; an isolated nucleic acid molecule having a nucleotide sequence having at least 95% identity to the nucleotide sequence set forth in SEQ ID NO:1 wherein said nucleotide sequence encodes a polypeptide capable of interacting with a Fzd4 receptor, such as the Wnt7b nucleotide sequence set forth in SEQ ID NO:4; a polypeptide having an amino acid sequence encoded by a nucleotide sequence set forth in SEQ ID NO:1 or SEQ ID NO:4; a polypeptide having an amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:3; a polypeptide having 95% identity to an amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:3 such as a polypeptide having the amino acid sequence set forth in SEQ ID NO:5 or SEQ ID NO:6; a fragment of an above described polypeptide or nucleotide sequence; or any small molecule, glycoprotein, peptidomimetic, lipid, antibody, ligand, sterol, steroid, hormone, kinase, kinase inhibitor, enzyme, enzyme inhibitor, carbohydrate, deaminase, deaminase inhibitor, G-protein, G-protein receptor inhibitor, calcium channel modulator, hormone receptor modulator, alcohol, phosphatase, or lactone capable of binding the Fzd4 receptor (Genbank NM--012193). Some Wnt7B-like molecules are described in U.S. Pat. No. 6,653,448 B1, herein incorporated by reference in its entirety. Particularly preferred Wnt7b-like molecules include Mus musculus Wnt7b (Genbank Acc. No. BC0066004), the nucleotide sequence of which is set forth in SEQ ID NO:1, the amino acid sequence of which is set forth in SEQ ID NO:2 and SEQ ID NO:3; Homo sapiens Wnt7b (Genbank Acc. No. AB062766), the nucleotide sequence of which is set forth in SEQ ID NO:4, the amino acid sequence of which is set forth in SEQ ID NO:5 and SEQ ID NO:6; and fragments and variants thereof. Methods of assessing receptor binding are known in the art and described elsewhere herein. Any method of assessing expression level known in the art may be used to assess myc or cyclin D expression. Methods of assessing expression levels are described elsewhere herein. Methods of assessing cell cycle stage are known in the art and described elsewhere herein.
[0029]By "Tie2 pathway repressor" is intended any Tie2 receptor antagonist including but not limited to, an Ang2-like molecule, or any Bad activator, Casp9 activator, or FH activator. By "Ang2-like molecule" is intended an isolated Ang2 nucleic acid molecule having the nucleotide sequence of set forth in SEQ ID NO:7 or having 95% identity to the nucleotide sequence set forth in SEQ ID NO:7; an isolated Ang2 polypeptide having the amino acid sequence set forth in SEQ ID NO:8 or SEQ ID NO:9 or having 95% identity to the amino acid sequence set forth in SEQ ID NO:8 or SEQ ID NO:9; an isolated Ang2 nucleic acid molecule having the nucleotide sequence of set forth in SEQ ID NO:10 or having 95% identity to the nucleotide sequence set forth in SEQ ID NO:10; an isolated Ang2 polypeptide having the amino acid sequence set forth in SEQ ID NO:11 or SEQ ID NO:12 or having 95% identity to the amino acid sequence set forth in SEQ ID NO:11 or SEQ ID NO:12; a fragment of an isolated Ang2 nucleic acid molecule encoding a polypeptide capable of interacting with an Ang2 receptor such as Tie2; a fragment of an isolated Ang2 polypeptide capable of interacting with an Ang2 receptor such as Tie2; or a small molecule, glycoprotein, peptidomimetic, lipid, antibody, ligand, sterol, steroid, hormone, kinase, kinase inhibitor, enzyme, enzyme inhibitor, carbohydrate, deaminase, deaminase inhibitor, alcohol, phosphatase, or lactone capable of interacting with Tie2 (Genbank No. AB086825). Particularly preferred Ang2-like molecules include Mus musculus Ang2 (Genbank Acc. No. NM007426), the nucleotide sequence of which is set forth in SEQ ID NO:7, the amino acid sequence of which is set forth in SEQ ID NO:8 and SEQ ID NO:9; Homo sapiens Ang2 (Genbank Acc. No. NM001147), the nucleotide sequence of which is set forth in SEQ ID NO:10, the amino acid sequence of which is set forth in SEQ ID NO:11 and SEQ ID NO:12; and fragments and variants thereof.
[0030]The invention encompasses isolated or substantially purified nucleic acid or protein compositions. An "isolated" or substantially "purified" nucleic acid molecule, polypeptide, or biologically active portion thereof, is substantially free of other cellular material or culture medium when produced by recombinant techniques or substantially free of chemical precursors or other chemicals when chemically synthesized. Preferably, an "isolated" nucleic acid molecule is free of sequences (preferably polypeptide encoding sequences) that naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived.
[0031]Fragments and variants of the Wnt7b and Ang2 nucleotide sequences and proteins encoded thereby are also encompassed by the present invention. By "fragment" is intended a portion of the nucleotide sequence or a portion of the amino acid sequence and hence protein encoded thereby. Fragments of a nucleotide sequence may encode protein fragments that retain the biological activity of the native protein and hence exhibit a Wnt7b or Ang2 activity. Alternatively, fragments of a nucleotide sequence that are useful as hybridization probes generally do not encode fragment proteins retaining biological activity. Thus, fragments of a nucleotide sequence may range from at least about 20 nucleotides, about 50 nucleotides, about 100 nucleotides, and up to the full-length nucleotide sequence encoding the proteins of the invention.
[0032]A fragment of a Wnt7b or an Ang2 nucleotide sequence that encodes a biologically active portion of a Wnt7b or an Ang2 protein of the invention will encode at least 15, 25, 30, 50, 100, 150, 200, 250, 300, 350 or 353 contiguous amino acids, or up to the total number of amino acids present in a full-length Wnt7b or at least 15, 25, 30, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 496 contiguous amino acids, or up to the total number of amino acids present in a full-length Ang2 protein of the invention. Fragments of a Wnt7b or an Ang2 nucleotide sequence that are useful as hybridization probes or PCR primers generally need not encode a biologically active portion of a Wnt7b or an Ang2 protein. Polypeptide fragments of particular interest include those comprising the receptor binding region of the polypeptide.
[0033]Thus, a fragment of a Wnt7b or an Ang2 nucleotide sequence may encode a biologically active portion of a Wnt7b or an Ang2 or it may be a fragment that can be used as a hybridization probe or PCR primer using methods disclosed below. A biologically active portion of a Wnt7b or an Ang2 can be prepared by isolating a portion of one of the Wnt7b or an Ang2 nucleotide sequences of the invention, expressing the encoded portion of the Wnt7b or an Ang2 protein (e.g., by recombinant expression in vitro), and assessing the activity of the encoded portion of the Wnt7b or an Ang2 protein. Nucleic acid molecules that are fragments of a Wnt7b nucleotide sequence comprise at least 16, 20, 50, 75, 100, 150, 200, 250, 300, 347, 350, 400, 450, 500, 550, 600, 650, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, or 2544 nucleotides, or up to the number of nucleotides present in a full-length Wnt7b nucleotide sequence disclosed herein. Nucleic acid molecules that are fragments of an Ang2 nucleotide sequence comprise at least 16, 20, 50, 75, 100, 150, 200, 250, 300, 347, 350, 400, 450, 500, 550, 600, 650, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, or 2475 nucleotides, or up to the number of nucleotides present in a full-length Ang2 nucleotide sequence disclosed herein.
[0034]By "variants" is intended substantially similar sequences. For nucleotide sequences, conservative variants include those sequences that, because of the degeneracy of the genetic code, encode the amino acid sequence of one of the Wnt7b or Ang2 polypeptides of the invention. Naturally occurring allelic variants such as these can be identified with the use of well-known molecular biology techniques, as, for example, with polymerase chain reaction (PCR) and hybridization techniques as outlined below. Variant nucleotide sequences also include synthetically derived nucleotide sequences, such as those generated, for example, by using site-directed mutagenesis [but which still encode a Wnt7b or an Ang2 protein of the invention]. Generally, variants of a particular nucleotide sequence of the invention will have at least about 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, and more preferably at least about 98%, 99% or more sequence identity to that particular nucleotide sequence as determined by sequence alignment programs described elsewhere herein using default parameters.
[0035]By "variant" protein is intended a protein derived from the native protein by deletion (so-called truncation) or addition of one or more amino acids to the N-terminal and/or C-terminal end of the native protein; deletion or addition of one or more amino acids at one or more sites in the native protein; or substitution of one or more amino acids at one or more sites in the native protein. Variant proteins encompassed by the present invention are biologically active, that is they continue to possess the desired biological activity of the native protein, that is, a Wnt7b or an Ang2 activity as described herein. Such variants may result from, for example, genetic polymorphism or from human manipulation. Biologically active variants of a native Wnt7b or an Ang2 protein of the invention will have at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, and more preferably at least about 98%, 99% or more sequence identity to the amino acid sequence for the native protein as determined by sequence alignment programs described elsewhere herein using default parameters. A biologically active variant of a protein of the invention may differ from that protein by as few as 1-15 amino acid residues, as few as 1-10, such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue. Biologically active variants of the polypeptide of the invention include Wnt7b polypeptides from other species such as H. sapiens (SEQ ID NOS:5 and 6) and Ang2 polypeptides from other species such as H. sapiens (SEQ ID NOS:11 and 12). Additional variants are discussed in Davis et al. (2003) Nature Structural Biology 10:38-44 and corrigendum p. 146, herein incorporated by reference in its entirety.
[0036]The proteins of the invention may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants of the Wnt7b or an Ang2 protein can be prepared by mutations in the DNA. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Kunkel (1985) Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al. (1987) Methods in Enzymol. 154:367-382; U.S. Pat. No. 4,873,192; Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York) and the references cited therein. Guidance as to appropriate amino acid substitutions that do not affect biological activity of the protein of interest may be found in the model of Dayhoff et al. (1978) Atlas of protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, D.C.), herein incorporated by reference. Conservative substitutions, such as exchanging one amino acid with another having similar properties, may be preferable.
[0037]Thus, the genes and nucleotide sequences of the invention include both the naturally occurring sequences as well as mutant forms. Likewise, the proteins of the invention encompass both naturally occurring proteins as well as variations and modified forms thereof. Such variants will continue to possess the desired Wnt7b or Ang2 activity. Obviously, the mutations that will be made in the DNA encoding the variant must not place the sequence out of reading frame and preferably will not create complementary regions that could produce secondary mRNA structure. See, EP Patent Application Publication No. 75,444.
[0038]The deletions, insertions, and substitutions of the protein sequence encompassed herein are not expected to produce radical changes in the characteristics of the protein. However, when it is difficult to predict the exact effect of the substitution, deletion, or insertion in advance of doing so, one skilled in the art will appreciate that the effect will be evaluated by routine screening assays. That is, the activity can be evaluated by Wnt7b or Ang2 assays such as, but not limited to, receptor binding assays, immunological assays, or secretion assays. See, for example, U.S. Pat. No. 6,653,448; PCT Publication WO 03/004045; Davis et al. (2002) Nature Structural Biology 10:44; and Robitaille et al. (2002) Nature Genetics 32:326-330; herein incorporated by reference in their entirety.
[0039]Variant nucleotide sequences and proteins also encompass sequences and proteins derived from a mutagenic and recombinogenic procedure such as DNA shuffling. With such a procedure, one or more different Wnt7b or Ang2 coding sequences can be manipulated to create a new Wnt7b or Ang2 possessing the desired properties. In this manner, libraries of recombinant polynucleotides are generated from a population of related sequence polynucleotides comprising sequence regions that have substantial sequence identity and can be homologously recombined in vitro or in vivo. For example, using this approach, sequence motifs encoding a domain of interest may be shuffled between the Wnt7b or an Ang2 gene of the invention and other known Wnt7b or an Ang2 genes to obtain a new gene coding for a protein with an altered property of interest e.g. receptor binding, agonism, or antagonism. Strategies for such DNA shuffling are known in the art. See, for example, Stemmer (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751; Stemmer (1994) Nature 370:389-391; Crameri et al. (1997) Nature Biotech. 15:436-438; Moore et al. (1997) J. Mol. Biol. 272:336-347; Zhang et al. (1997) Proc. Natl. Acad. Sci. USA 94:4504-4509; Crameri et al. (1998) Nature 391:288-291; and U.S. Pat. Nos. 5,605,793 and 5,837,458.
[0040]The following terms are used to describe the sequence relationships between two or more nucleic acids or polynucleotides: (a) "reference sequence", (b) "comparison window", (c) "sequence identity", (d) "percentage of sequence identity", and (e) "substantial identity".
[0041](a) As used herein, "reference sequence" is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset or the entirety of a specified sequence; for example, as a segment of a full-length cDNA or gene sequence or the complete cDNA or gene sequence.
[0042](b) As used herein "comparison window" makes reference to a contiguous and specified segment of a polynucleotide sequence, wherein the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e. gaps) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Generally the comparison window is at least 20 contiguous nucleotides in length, and optionally can be 30, 40, 50, 100, or longer. Those of skill in the art understand that to avoid a high similarity to a reference sequence due to inclusion of gaps in the polynucleotide sequence a gap penalty is typically introduced and is subtracted from the number of matches.
[0043]Methods of alignment of sequences for comparison are well known in the art. Thus, the determination of percent sequence identity between any two sequences can be accomplished using a mathematical algorithm. Computer implementations of these mathematical algorithms can be utilized for comparison of sequences to determine sequence identity. For purposes of the present invention, comparison of nucleotide or protein sequences for determination of percent sequence identity to the sequences disclosed herein is preferably made using the Multiple Sequence Alignment V2.0: http://xylian.igh.cnrs.fr/msa/msa.html with its default parameters or any equivalent program. By "equivalent program" is intended any sequence comparison program that, for any two sequences in question, generates an alignment having identical nucleotide or amino acid residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by the preferred program.
[0044](c) As used herein, "sequence identity" or "identity" in the context of two nucleic acid or polypeptide sequences makes reference to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. When sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have "sequence similarity" or "similarity". Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, Calif.).
[0045](d) As used herein, "percentage of sequence identity" means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity.
[0046](e)(i) The term "substantial identity" of polynucleotide sequences means that a polynucleotide comprises a sequence that has at least 70% sequence identity, preferably at least 80%, more preferably at least 90%, and most preferably at least 95%, compared to a reference sequence using one of the alignment programs described using standard parameters. One of skill in the art will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning, and the like. Substantial identity of amino acid sequences for these purposes normally means sequence identity of at least 60%, more preferably at least 70%, 80%, 90%, and most preferably at least 95%.
[0047]Another indication that nucleotide sequences are substantially complementary is if two molecules hybridize to each other under stringent conditions. Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. However, stringent conditions encompass temperatures in the range of about 1° C. to about 20° C. lower than the Tm, depending upon the desired degree of stringency as otherwise qualified herein. Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides they encode are substantially identical. This may occur, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. One indication that two nucleic acid sequences are substantially identical is when the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the polypeptide encoded by the second nucleic acid.
[0048](e)(ii) The term "substantial identity" in the context of a peptide indicates that a peptide comprises a sequence with at least 70% sequence identity to a reference sequence, preferably 80%, more preferably 85%, most preferably at least 90% or 95% sequence identity to the reference sequence over a specified comparison window. Preferably, optimal alignment is conducted using the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453. An indication that two peptide sequences are substantially identical is that one peptide is immunologically reactive with antibodies raised against the second peptide. Thus, a peptide is substantially identical to a second peptide, for example, where the two peptides differ only by a conservative substitution. Peptides that are "substantially similar" share sequences as noted above except that residue positions that are not identical may differ by conservative amino acid changes.
[0049]Methods of assaying interaction of a compound with an Ang2 receptor such as Tie2 or a Wnt7b receptor such as Fzd4 are known in the art. Any method known in the art for assessing protein-protein interaction such as, but not limited to, SPR, immunoprecipitation, co-immunoprecipitation, receptor binding assays, yeast 2-hybrid, phosphorylation assays, dephosphorylation assays, Western blots, ELISA, cross-linking experiments, chemiluminescence, silver staining, enzymatic assays, ponceau S staining, multiplex RT-PCR, immunohistochemical assays, radioimmunoassay, colorimetric analysis, immunoradiometric assays, immunochemistry, positron emission tomography, Northern blotting, fluorometric assays, fluorescence activated cell sorter staining of permeabilized cells, and radioimmunosorbent assays, is suitable for use in the invention. See for example, Davis et al. (2003) Nature Structural Biology 10:38-44; Walker, John, ed. (2002)Protein Protocols on CD-ROM v. 2; and Ausubel et al., eds. (1995) Current Protocols in Molecular Biology, (Greene Publishing and Wiley-Interscience, New York); WO 03/004045; Robitaille et al. (2002) Nature 32:326-330; herein incorporated by reference in their entirety.
[0050]A "Wnt7b expression modulating agent" is a compound or agent that results in an altered Wnt7b expression level compared to the Wnt7b mRNA or protein expression level observed in the absence of the compound. Wnt7b expression modulating agents can be identified in a method wherein a cell is contacted with a candidate compound and the expression of Wnt7b mRNA or protein in the cell is determined. The level of expression of Wnt7b mRNA or protein in the presence of the compound is compared to the Wnt7b mRNA or protein expression level in the absence of the candidate compound. The candidate compound can then be identified as a modulator of Wnt-7b expression based on this comparison. For example, when Wnt7b mRNA or protein expression levels are significantly greater in the presence of the compound than its absence, the candidate compound is identified as a stimulator of Wnt7b expression. Alternatively when expression of Wnt7b is significantly less in the absence of the compound than in its presence, the candidate compound is identified as an inhibitor of Wnt7b expression. The level of Wnt7b mRNA or protein expression in the cells can be determined by expression level assays described elsewhere herein. A significant increase in expression level is an increase in the range of 1% to 1000%, particularly 5% to 500%, more particularly 5% to 250%, yet more particularly 10% to 100%. A significant decrease in expression level is a decrease in the range of 1% to 100%, particularly 5% to 100%, more particularly 10% to 100%, yet more particularly 20% to 100%, yet still more particularly 30% to 100%, even yet still more particularly 50% to 100%.
[0051]Methods of determining expression levels are known in the art and include, but are not limited to, qualitative Western blot analysis, immunoprecipitation, radiological assays, polypeptide purification, spectrophotometric analysis, Coomassie staining of acrylamide gels, ELISAs, RT-PCR, 2-D gel electrophoresis, microarray analysis, in situ hybridization, chemiluminescence, silver staining, enzymatic assays, ponceau S staining, multiplex RT-PCR, immunohistochemical assays, radioimmunoassay, colorimetric analysis, immunoradiometric assays, positron emission tomography, Northern blotting, fluorometric assays and SAGE. See, for example, Ausubel et al, eds. (2002) Current Protocols in Molecular Biology, Wiley-Interscience, New York, N.Y.; Coligan et al (2002) Current Protocols in Protein Science, Wiley-Interscience, New York, N.Y.; and Sun et al. (2001) Gene Ther. 8:1572-1579, herein incorporated by reference.
[0052]Methods of determining cell cycle stage are known in the art and include, but are not limited to, flow cytometry, centrifugal elutriation, immunofluorescence, BrdU incorporation, cyclin dependent kinase activity assays, propidium iodide staining, DAPI staining, Hoechst 33342 staining, cyclin D expression analysis, cyclin E expression analysis, cyclin A expression analysis, cyclin B1 expression analysis, tritiated thymidine incorporation, and counterflow centrifugal elutriation (Current Protocols in Cell Biology (2003) John Wiley & Sons; herein incorporated by reference in its entirety).
[0053]By "angiogenesis" is intended the process of vascularization of a tissue involving the development of capillary blood vessels or vascular endothelial cell vessels. The process of angiogenesis encompasses the metabolically controlled regression of capillary blood vessels. Examples of naturally occurring metabolically controlled regression of capillary blood vessels include, but are not limited to, regression during ocular development and regression after a local immune or inflammatory response. By "vascular endothelial cell vessel" is intended any vessel or tube comprised primarily of vascular endothelial cells, often also comprised of additional components such as a basal lamina. Vascular endothelial cell vessels include those formed in vivo or in vitro. The length, diameter, and continuity of a vascular endothelial cell vessel ranges. See Frietas Jr., R. A. (1999) Nanomedicine, Volume I: Basic Capabilities Landes Bioscience Georgetown Tex. and Gray, Henry. Anatomy of the Human Body. Philadelphia: Lea & Febiger, 1918; Bartleby.com, 2000. www.bartleby.com/107/. 9/8/05, Section V Angiology; herein incorporated by reference in their entirety. By "vascular endothelial cell" is intended a simple squamous epithelial cell such as those comprising the simple squamous epithelial tissue lining the blood vessels.
[0054]By "angiogenic-related disorder" is intended any disorder, disease, or developmental condition that involves atypical growth, extension, retention, development, progression, increase, or persistence of a blood vessel such as, but not limited to, a capillary blood vessel, wherein the majority of cells in said blood vessel are vascular endothelial cells. Angiogenic related disorders include, but are not limited to, angiomas, capillary angiomas, hemangioma, capillary angioma, angioma simplex, angioma cavernosum, cherry angioma, senile angioma, serpigninous angioma, spider angioma, telangiectactic angioma, angioma venosum racemosum, angiomalacia, angiomatosis, angiomegaly, angiomyoma, angiomyosarcoma, angiomyoneuroma, angiosarcoma, angioscotoma, angiosis, angiotelectasis, angiotitis, vasculitis, Wegener's granulomatosis, Henoch-Schonlein purpura, microscopic polyangiitis, hemangioma, hypersensitivity vasculitis, erythema nodosum, arteriovenous fistulas, damage response, irritant response, ocular angiogenic disorders, hypoxia response, vascular tumor, and hyperproliferative disorders such as, but not limited to, vascularized tumors, cancers, and neoplasms.
[0055]By "vascular endothelial cell vessel-related disorder" is intended any disorder or disease involving a tissue comprising a vascular endothelial cell vessel wherein the tissue exhibits a disorder; of particular interest are developmental disorders and hyperproliferative disorders.
[0056]The terms "cancer" or "neoplasms" include malignancies of the various organ systems, such as those affecting the lung, breast, thyroid, lymphoid, gastrointestinal, or genito-urinary tract, as well as adenocarcinomas which include malignancies such as most colon cancers, renal-cell carcinoma, prostate cancer and/or testicular tumors, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus.
[0057]The term "carcinoma" is art recognized and refers to malignancies of epithelial or endocrine tissues including respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostatic carcinomas, endocrine system carcinomas, and melanomas. Exemplary carcinomas include those forming from tissue of the cervix, lung, prostate, breast, head and neck, colon and ovary. The term also includes carcinosarcomas, e.g., which include malignant tumors composed of carcinomatous and sarcomatous tissues. An "adenocarcinoma" refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures.
[0058]The term "sarcoma" is art recognized and refers to malignant tumors of mesenchymal derivation.
[0059]Tumors and cancers of the blood vessels include, but are not limited to hemangioma, lymphangioma, glomus tumor (glomangioma), vascular ectasias, and bacillary angiomatosis, and intermediate-grade (borderline low-grade malignant) tumors, such as Kaposi sarcoma and hemangioendothelioma, and malignant tumors, such as angiosarcoma and hemangiopericytoma.
[0060]Tumors and cancers of the skin include, but are not limited to, malignant melanoma; benign epithelial tumors, including but not limited to, seborrheic keratoses, acanthosis nigricans, fibroepithelial polyp, epithelial cyst, keratoacanthoma, and adnexal (appendage) tumors; premalignant and malignant epidermal tumors, including but not limited to, actinic keratosis, squamous cell carcinoma, basal cell carcinoma, and merkel cell carcinoma; tumors of the dermis, including but not limited to, benign fibrous histiocytoma, dermatofibrosarcoma protuberans, xanthomas, and dermal vascular tumors; tumors of cellular immigrants to the skin, including but not limited to, histiocytosis X, mycosis fungoides (cutaneous T-cell lymphoma), and mastocytosis.
[0061]Tumors and cancers of cells found in the bone marrow include, but are not limited to, disorders arising from these cells. These disorders include but are not limited to the following: diseases involving hematopoietic stem cells; committed lymphoid progenitor cells; lymphoid cells including B and T-cells; committed myeloid progenitors, including monocytes, granulocytes, and megakaryocytes; and committed erythroid progenitors. These include but are not limited to the leukemias, including B-lymphoid leukemias, T-lymphoid leukemias, undifferentiated leukemias; erythroleukemia, megakaryoblastic leukemia, monocytic; [leukemias are encompassed with and without differentiation]; chronic and acute lymphoblastic leukemia, chronic and acute lymphocytic leukemia, chronic and acute myelogenous leukemia, lymphoma, myelo dysplastic syndrome, chronic and acute myeloid leukemia, myelomonocytic leukemia; chronic and acute myeloblastic leukemia, chronic and acute myelogenous leukemia, chronic and acute promyelocytic leukemia, chronic and acute myelocytic leukemia, hematologic malignancies of monocyte-macrophage lineage, such as juvenile chronic myelogenous leukemia; secondary AML, antecedent hematological disorder; reactive cutaneous angioendotheliomatosis; fibrosing disorders involving altered expression in dendritic cells, disorders including systemic sclerosis, E-M syndrome, epidemic toxic oil syndrome, eosinophilic fasciitis localized forms of scleroderma, keloid, and fibrosing colonopathy; angiomatoid malignant fibrous histiocytoma; carcinoma, including primary head and neck squamous cell carcinoma; sarcoma, including Kaposi's sarcoma; fibroadenoma and phyllodes tumors, including mammary fibroadenoma; stromal tumors; phyllodes tumors, including histiocytoma; T-cell lymphomas; and B-cell lymphomas.
[0062]Tumors and cancers of the heart include, but are not limited to, primary cardiac tumors, such as myxoma, lipoma, papillary fibroelastoma, rhabdomyoma, and sarcoma, and cardiac effects of noncardiac neoplasms.
[0063]Tumors and cancers of the B-cells include, but are not limited to precursor B-cell neoplasms, such as lymphoblastic leukemia/lymphoma. Peripheral B-cell neoplasms include, but are not limited to, chronic lymphocytic leukemia/small lymphocytic lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, Burkitt lymphoma, plasma cell neoplasms, multiple myeloma, and related entities, lymphoplasmacytic lymphoma (Waldenstrom macroglobulinemia), mantle cell lymphoma, marginal zone lymphoma (MALToma), and hairy cell leukemia.
[0064]Tumors and cancers of the liver include, but are not limited to nodular hyperplasias, adenomas, and malignant tumors, including primary carcinoma of the liver and metastatic tumors.
[0065]Tumors and cancers of the brain include, but are not limited to gliomas, including astrocytoma, including fibrillary (diffuse) astrocytoma and glioblastoma multiforme, pilocystic astrocytoma, pleomorphic xanthoastrocytoma, and brain stem glioma, oligodendroglioma, and ependymoma and related paraventricular mass lesions, neuronal tumors, poorly differentiated neoplasms, including medulloblastoma, other parenchymal tumors, including primary brain lymphoma, germ cell tumors, and pineal parenchymal tumors, meningiomas, metastatic tumors, paraneoplastic syndromes, peripheral nerve sheath tumors, including schwannoma, neurofibroma, and malignant peripheral nerve sheath tumor (malignant schwannoma), and neurocutaneous syndromes (phakomatoses), including neurofibromatosis, including Type 1 neurofibromatosis (NF1) and TYPE 2 neurofibromatosis (NF2), tuberous sclerosis, and Von Hippel-Lindau disease.
[0066]Tumors and cancers of the ovary include, but are not limited to, ovarian tumors such as, tumors of coelomic epithelium, serous tumors, mucinous tumors, endometrioid tumors, clear cell adenocarcinoma, cystadenofibroma, Brenner tumor, surface epithelial tumors; germ cell tumors such as mature (benign) teratomas, monodermal teratomas, immature malignant teratomas, dysgerminoma, endodermal sinus tumor, choriocarcinoma; sex cord-stomal tumors such as, granulosa-theca cell tumors, thecoma-fibromas, androblastomas, hill cell tumors, and gonadoblastoma; and metastatic tumors such as Krukenberg tumors.
[0067]Tumors and cancers of the kidney include, but are not limited to, benign tumors, such as renal papillary adenoma, renal fibroma or hamartoma (renomedullary interstitial cell tumor), angiomyolipoma, and oncocytoma, and malignant tumors, including renal cell carcinoma (hypemephroma, adenocarcinoma of kidney), which includes urothelial carcinomas of renal pelvis.
[0068]Tumors and cancers of the skeletal muscle include, but are not limited to, rhabdomyosarcoma.
[0069]Tumors and cancers of the bone-forming cells include, but are not limited to, osteoma, osteoid osteoma, osteoblastoma, osteosarcoma, osteochondroma, chondromas, chondroblastoma, chondromyxoid fibroma, chondrosarcoma, fibrous cortical defects, fibrous dysplasia, fibrosarcoma, malignant fibrous histiocytoma, Ewing sarcoma, primitive neuroectodermal tumor, giant cell tumor, and metastatic tumors.
[0070]Tumors and cancers of the pancreas include, but are not limited to, cystic tumors and carcinoma of the pancreas; islet cell tumors, including but not limited to, insulinomas, gastrinomas, and other rare islet cell tumors.
[0071]Tumors and cancers of the breast include, but are not limited to, stromal tumors such as fibroadenoma, phyllodes tumor, and sarcomas, and epithelial tumors such as large duct papilloma; carcinoma of the breast including in situ (noninvasive) carcinoma that includes ductal carcinoma in situ (including Paget's disease) and lobular carcinoma in situ, and invasive (infiltrating) carcinoma including, but not limited to, invasive ductal carcinoma, no special type, invasive lobular carcinoma, medullary carcinoma, colloid (mucinous) carcinoma, tubular carcinoma, and invasive papillary carcinoma, and miscellaneous malignant neoplasms.
[0072]Tumors and cancers of the male breast include, but are not limited to, carcinoma.
[0073]Tumors and cancers of the prostate include, but are not limited to, carcinoma.
[0074]Tumors and cancers of the colon include, but are not limited to, non-neoplastic polyps, adenomas, familial syndromes, colorectal carcinogenesis, colorectal carcinoma, and carcinoid tumors.
[0075]Tumors and cancers of the lung include, but are not limited to, bronchogenic carcinoma, including paraneoplastic syndromes, bronchioloalveolar carcinoma, neuroendocrine tumors, such as bronchial carcinoid, miscellaneous tumors, and metastatic tumors; pleural tumors, including solitary fibrous tumors (pleural fibroma) and malignant mesothelioma.
[0076]Tumors and cancers of the thymus include, but are not limited to, thymomas, including germ cell tumors, lymphomas, Hodgkin disease, and carcinoids. Thymomas can include benign or encapsulated thymoma, and malignant thymoma Type I (invasive thymoma) or Type II, designated thymic carcinoma.
[0077]Tumors and cancers of the tonsils include, but are not limited to, non-Hodgkin's lymphoma and B-cell lymphoma.
[0078]The invention provides compositions with a vascular endothelial cell modulating activity and methods of modulating angiogenesis, angiogenic-related disorders, a vascular endothelial cell vessel, vascular endothelial cell vessel-related disorders, capillary bed development, and apoptosis of a vascular endothelial cell. By "modulating" is intended a change of at least 1%, 5%, preferably 10%, 20%, more preferably 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99% or more in the volume or number of vascular endothelial cells in a target area or a change in the cell cycle stage of a vascular endothelial cell. Modulation of vascular endothelial cell vessels may be evaluated by any means known in the art including but not limited to those methods described elsewhere herein. In an embodiment a vascular endothelial cell modulating composition of the invention causes a vascular endothelial cell vessel to regress, diminish, shrink, retreat, disappear, reverse, leave, depart, retract, or decrease. Such a regression may result from apoptosis of one or more vascular endothelial cells constituting the vascular endothelial cell vessel of interest.
[0079]By "apoptosis" or "programmed cell death" is intended a normal physiological process requiring regulated metabolic activity by the dying cell often characterized by fragmentation of the genomic DNA or other physiological alterations. Physiological alterations of the cell associated with apoptosis include, but are not limited to, DNA fragmentation, MC540 incorporation, annexin V-FITC binding, propidium iodide (PI) labeling of hypodiploid nuclei, lactate dehydrogenase (LDH) release, caspase-3 activation, presence of cyclin D1, cytochrome c leakage from the mitochondria, BAX cellular redistribution, cleavage of poly (ADP-ribose) polymerase (PARP) to an 85-kDa apoptotic fragment, dissipation of the mitochondrial transmembrane potential, phosphatidylserine exposure, tissue transglutaminase activation, cell shrinkage, chromatin condensation, nuclear fragmentation, loss of membrane integrity, blebbing of plasma membranes, loss of membrane integrity, or cell membrane permeability to PI. Apoptosis can be assessed by any method of assessing an apoptotic characteristic known in the art such as, but not limited to, a colorimetric assay, propidium iodide labeling, flow cytometry, TUNEL, ISEL, rhodamine 123 fluorescence, DIOC6(3) fluorescence, immunocytochemical detection by Zenon technology, FLICAs, PARP cleavage assays, detergent resistance assays, and cadaverine binding (Current Protocols in Cell Biology (2003) John Wiley & Sons; herein incorporated by reference in its entirety).
[0080]An agent of the invention comprising a Wnt pathway stimulator and a Tie2 pathway repressor (also referred to herein as "active compounds") can be incorporated into pharmaceutical compositions suitable for administration to a subject. Such compositions typically comprise a Wnt pathway stimulator and a Tie2 pathway repressor, and a pharmaceutically acceptable carrier.
[0081]By "subject" is intended a mammal, e.g., a human, or an experimental or animal or disease model or mammalian tissue or mammalian cells. Suitable subjects include mammals, particularly humans, at risk for an angiogenic-related disorder, particularly a vascular endothelial cell related disorder; mammals, particularly humans, exhibiting an angiogenic-related disorder, such as but not limited to a vascular endothelial cell related disorder; tissue obtained from a mammal exhibiting an angiogenic-related disorder; cells obtained from a mammal exhibiting an angiogenic-related disorder; cells cultured from a mammal exhibiting an angiogenic-related disorder; and mammals, particularly humans, at risk for a vascular endothelial cell vessel related disorder. The subject can also be a non-human mammal such as, but not limited to, a horse, hamster, guinea pig, mouse, rabbit, dog, pig, goat, cow, rat, monkey, chimpanzee, sheep, or other domestic animal. Subjects exhibiting an angiogenic-related disorder can be identified by any means known in the art including, but not limited to, physical examination, visual observation, angiogram, familial history analysis, ultrasound, MRI, bone density scan, and retinal scan.
[0082]The term "administer" is used in its broadest sense and includes any method of introducing the compositions of the present invention into a subject. By "subject" is intended a mammal, e.g., a human, or an experimental or animal or disease model. The subject can also be a non-human animal such as, but not limited to, a non-human primate, horse, cow, goat, pig, rabbit, mouse, guinea pig, dog, or other domestic animal. Additionally the compositions of the invention find use in the treatment of disorders described herein. Thus, therapies for angiogenic related disorders or disorders with associated capillary bed development are encompassed herein. "Treatment" is herein defined as the application or administration of an agent of the invention to a patient, or application or administration of an agent of the invention to an isolated tissue or cell line from a patient, who has a disease or symptom of a disease, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disease or symptoms of the disease.
[0083]As used herein the language "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
[0084]A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, intraperitoneal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic.
[0085]Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL® (BASF; Parsippany, N.J.), or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride, in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.
[0086]Sterile injectable solutions can be prepared by incorporating the active compounds (e.g., a Wnt pathway activator component and a Tie2 pathway repressor component) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying, which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
[0087]Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth, or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring. For administration by inhalation, the compounds are delivered in the form of an aerosol spray from a pressurized container or dispenser that contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
[0088]In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.
[0089]It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated with each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. Depending on the type and severity of the disease, about 0.1 μg/kg to about 1500 mg/kg of an agent of the invention is an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. A typical daily dosage might range from about 1 μg/kg to about 100 mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays. An exemplary dosing regimen is disclosed in WO 94/04188. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
[0090]Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art. The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
[0091]The vascular endothelial cell modulating compounds described herein can be administered transdermally. Transdermal administration typically involves the delivery of a pharmaceutical agent for percutaneous passage of the drug into the systemic circulation of the subject or patient. The skin sites include anatomic regions for transdermally administering the drug and include the forearm, abdomen, chest, back, buttock, mastoidal area, and the like.
[0092]Other items may be contained in the device, such as other pharmaceutically acceptable carriers, depending on the desired device characteristics. For example, the compositions according to this invention may also include one or more preservatives or bacteriostatic agents, e.g., methyl hydroxybenzoate, propyl hydroxybenzoate, chlorocresol, benzalkonium chlorides, and the like. These pharmaceutical compositions also can contain other active ingredients such as anti-microbial agents, particularly antibiotics, anesthetics, and antipruritic agents.
[0093]Another aspect of this invention provides for the topical delivery of an agent of the invention. This treatment regimen is suitable either for the systemic administration of the agent or for localized therapy, i.e., directly to pathological or diseased tissue.
[0094]Typically, the topical formulations will comprise a preparation for delivering the agent directly to the affected area comprising the complex, typically in concentrations in the range of from about 0.001% to 10%; preferably, from about 0.01 to about 10%; more preferably from about 0.1 to about 5%; and most preferably from about 1 to about 5%, together with a non-toxic, pharmaceutically acceptable topical carrier (Barry (eds). Dermatological Formulations: Percutaneous Absorption (1983) Marcel Dekker, Inc; for standard dosages of conventional pharmaceutical agents see, e.g., Physicians Desk Reference (1992 Edition); and American Medical Association (1992) Drug Evaluations Subscriptions).
[0095]Topical preparations can be prepared by combining the agent with conventional pharmaceutical diluents and carriers commonly used in topical dry, liquid, cream, and aerosol formulations. Ointment and creams may, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling substances. Such bases may include water and/or an oil such as liquid paraffin or a vegetable oil such as peanut oil or castor oil. Thickening agents which may be used according to the nature of the base include soft paraffin, aluminum stearate, cetostearyl alcohol, propylene glycol, polyethylene glycols, wool fat, hydrogenated lanolin, beeswax, and the like. Lotions may be formulated with an aqueous or oily base and will, in general, also include one or more of the following: stabilizing agents, emulsifying agents, dispersing agents, suspending agents, thickening agents, coloring agents, perfumes, and the like. Powders may be formed with the aid of any suitable base, e.g., talc, lactose, starch, and the like. Drops may be formulated with an aqueous base or non-aqueous base also comprising one or more dispersing agents, suspending agents, solubilizing agents, and the like.
[0096]Dosage forms for the topical administration of an agent of this invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants which may be required.
[0097]The ointments, pastes, creams, and gels also may contain excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, talc, and zinc oxide, or mixtures thereof. Powders and sprays also can contain excipients such as lactose, talc, aluminum hydroxide, calcium silicates, and polyamide powder, or mixtures of those substances. Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons such as butane and propane.
[0098]The methods of the present invention are also applicable to the delivery of pharmaceutical agents through mucosal membranes such as a gastrointestinal, sublingual, buccal, nasal, pulmonary, vaginal, corneal, and ocular membranes (Mackay et al. (1991) Adv. Drug Del. Rev. 7:313-338).
[0099]For delivery to the buccal or sublingual membranes, typically an oral formulation such as a lozenge, tablet, or capsule will be used. The method of manufacture of these formulations are known in the art, including, but not limited to, the addition of the agent to a pre-manufactured tablet; cold compression of an inert filler, a binder, and encapsulation.
[0100]Another oral formulation is one that can be applied with an adhesive such as the cellulose derivative, hydroxypropyl cellulose, to the oral mucosa, for example as described in U.S. Pat. No. 4,940,587, incorporated by reference. This buccal adhesive formulation, when applied to the buccal mucosa, allows for the controlled release of an agent into the mouth and through the buccal mucosa.
[0101]For delivery to the nasal and/or pulmonary membranes, typically an aerosol formulation will be employed. The term "aerosol" includes any gas-borne suspended phase of an agent of the invention which is capable of being inhaled into the bronchioles or nasal passages. Specifically, aerosol includes a gas-borne suspension of droplets of the compounds of the instant invention, as may be produced in a metered dose inhaler or nebulizer, or in a mist sprayer. Aerosol also includes a dry powder composition of the agent suspended in air or other carrier gas, which may be delivered by inhalation from an inhaler device.
[0102]The compositions of the invention are useful to treat any of the disorders discussed herein. The compositions are provided in therapeutically effective amounts. By "therapeutically effective amounts" is intended an amount sufficient to modulate the desired response.
[0103]The skilled artisan will appreciate that certain factors may influence the dosage required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a compound of the invention can include a single treatment or, preferably, can include a series of treatments. In a preferred example, a subject is treated with a therapeutically effective amount of the agent one time per week for between about 1 to 10 weeks, preferably between 2 to 8 weeks, more preferably between about 3 to 7 weeks, and even more preferably for about 4, 5, or 6 weeks. It will also be appreciated that the effective dosage used for treatment may increase or decrease over the course of a particular treatment. Changes in dosage may result and become apparent from the results of diagnostic assays as described herein.
[0104]Where a subject undergoing therapy exhibits a partial response or a relapse following a prolonged period of remission, subsequent course of treatment with an agent of the invention may be administered. Thus, subsequent to a period of time off from a first treatment period, which may have comprised a single dosing regimen or a multiple dosing regimen, a subject may receive one or more additional treatment periods comprising single or multiple dosing regimens. Such a period of time off between treatment periods is referred to herein as a time period of discontinuance. It recognized that the length of the time period of discontinuance is dependent upon the degree of disorder response achieved with any prior treatment periods with the vascular endothelial cell modulating compounds of the invention.
[0105]The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.
[0106]The results of treatment of a disorder may be assayed by any method known to one skilled in the art including, but not limited to, physical examination, laboratory, nuclear, and radiographic studies (i.e. computer tomography and/or magnetic resonance imagery), angiogram, angiography, ultrasound and other procedures.
[0107]In an embodiment, the invention provides a compound suitable for use as a cosmetic agent. A cosmetic agent of the invention may further comprise a delivery component such as an ointment or a lotion. A cosmetic agent of the invention finds use in the modulation, reduction, or diminishment of skin lesions characterized by visible capillary bed development or capillary bed persistence. Administration of a cosmetic agent of the invention occurs at a frequency that ranges from hourly to once per decade, particularly daily to annually, more particularly daily to weekly. Administration of a cosmetic agent of the invention may occur on a single occasion or on multiple occasions.
[0108]The following examples are offered by way of illustration and not limitation.
EXPERIMENTAL
Example 1
Mouse Breeding and Genotyping
[0109]Genotyping of Lrp5.sup.lacZ/lacZ, Lef1.sup.-/-, PU.1.sup.-/-, and TOPGAL mice was performed as previously described. When Lef1-mutant mice were produced as C57BL6/129 Sv F1 hybrids, they showed enhanced survival that allowed the analysis of hyaloid vessel regression postnatally. See McKercher et al (1996) EMBO J. 15:5647-5658; Kato et al. (2002) J. Cell Biol. 157:303-314; van Genderen et al. (1994) Genes Dev. 8:2691-2703; and DasGupta & Fuchs (1999) Development 126:4557-4568, herein incorporated by reference in their entirety.
Example 2
Dissections, Immunostaining, and Imaging
[0110]Hyaloid vessel preparation was performed as previously described with the exception that 5% (w/v) gelatin was used at 56° C. and allowed to set on ice before completion of the dissection. X-gal staining was done according to standard protocols. Indirect immunofluorescent staining and BrdU labeling was performed as previously described. Primary antibodies used were anti-vascular endothelial cell cadherin (Santa Cruz), anti-BrdU (Dako), and anti-F4/80 (Caltag Laboratories), all at 1:100 dilution. Secondary antibodies labeled with Alexa fluorochromes (Molecular Probes) were used at a 1:500 dilution. TUNEL labeling of apoptotic cells was performed using an in situ cell death detection kit (Roche).
Example 3
Plasmids
[0111]All Wnt complementary DNAs used were mouse. Wnt7b was expressed from pCDNA3 as a V5 and His-epitope tagged fusion protein. In addition, Fzd4m1 the sequence encoding the cysteine-rich domain (residues 54-QNLGYNV-60) was modified to encode AALAYAA. In Fzd4m2 the sequence encoding 105-MCT-107 was modified to encode ACA. The sequence alterations were made using a PCR-based mutagenesis strategy. The Fzd4m1 and Fzd4m2 were generated in pCDNA3. These expression plasmids were used in the paracrine and range-of-action signaling assays.
Example 4
Laser Capture Microdissections
[0112]Hyaloid macrophages were isolated from whole-mount hyaloid vessel preparations using the PixCell II laser-capture microdissection system (Arcturus). Macrophages were lifted from preparation on the laser-melted polymer membrane.
Example 5
Macrophage Rescue
[0113]Mouse bone marrow macrophages were isolated as previously described (Diez-Roux & Lang (1997) Development 124:3633-3638, herein incorporated by reference. Approximately 4000 macrophages from wild-type or Wnt7b.sup.d1/d1 animals were injected into the vitreous at P1 in a volume of 200 nl using a modification of a previously described trans-corneal technique. Animals were sacrificed at P8 and hyaloid vessels dissected.
Example 6
Luciferase and Range of Action Assays
[0114]For the Frizzled receptor activity screen SuperTOPFLASH cells carrying a reporter plasmid with Lef/Tcf binding sites and a minimal promoter upstream of an open reading frame encoding luciferase were plated into 60 mM dishes. The cells were transfected after 24 hours with combinations of 1 μg Lrp5-FLAG, 1 μg Wnt7b-V5H is, and 0.1 μg Fzd plasmid made up to 4 μg with pIRES-GFP using Fugene 6 (Roche). Forty-eight hours after transfection, cells were washed with PBS and luciferase activity was measured with a luciferase assay kit (Promega).
[0115]For the mixed cell paracrine signaling assay, 293 cells were transfected with plasmids expressing Wnt pathway ligands and overlaid on SuperTOPFLASH responder cells that were separately transfected with plasmids carrying LRP5 and a FZD4 receptor.
[0116]Wnt7b range of action experiments were carried out as described in Xu et al. (2004) Cell 116:883-895, herein incorporated by reference.
Example 8
Statistical Analysis
[0117]Vessel number was quantified using established methods (Huelskin and Birchmeier (2001) Curr. Opin. Genet. Dev. 11:547-553, herein incorporated by reference in its entirety). At least four hyaloid vessel preparations were quantified for each experiment. The student's T-test and ANOVA was used to assess statistical significance.
[0118]All publications, patents, and patent applications mentioned in the specification are indicative of the level of those skilled in the art to which this invention pertains. All publications, patents, and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application were specifically and individually incorporated by reference.
[0119]Having described the invention with reference to the exemplary embodiments, it is to be understood that it is not intended that any limitations or elements describing the exemplary embodiment set forth herein are to be incorporated into the meanings of the patent claims unless such limitations or elements are explicitly listed in the claims. Likewise, it is to be understood that it is not necessary to meet any or all of the identified advantages or objects of the invention disclosed herein in order to fall within the scope of any claims, since the invention is defined by the claims and since inherent and/or unforeseen advantages of the present invention may exist even though they may not be explicitly discussed herein.
[0120]Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.
Sequence CWU
1
1212544DNAMus musculusCDS(481)...(1542)Wnt7b 1gggtatcccg gccaaggcaa
cttgggcaaa ccctgggggc ggcacccgcc gcacactctg 60gtcaacctcc cctttaaaaa
aaacccgccc gcggaggagg cggatgtagg ggcggcctgt 120ccaatggcag ctgcggcgct
ttgggagact tggagacttg agccagagtg agcgacagaa 180ccggttcgca ccgacagacg
gacagaggac cagacagcca ctaaggagcg cttactgccc 240ccctccgggc ccctgccccg
aactccagcc ccagcgcctg ttactgcccc agatacagca 300agatgcgcgg tcctggcagc
gagacacggg cgagcactgt cccccggtcc ccgagccctg 360gcccctagcg cccagcgctg
ctgccctgca tcagggaggg ccgcggagac cccagcctca 420gttggcgcag gagccctgcg
ggtggggcct gcccagccca gccaggcgcg ccagcccacc 480atg ctc ctc ctg tcg ccg
cgc agc gcg ctg gtc tcc gtc tat tgc ccg 528Met Leu Leu Leu Ser Pro
Arg Ser Ala Leu Val Ser Val Tyr Cys Pro1 5
10 15cag atc ttt ctc ctt ctg tcc agc ggc agt tac cta
gca ttg tca tcc 576Gln Ile Phe Leu Leu Leu Ser Ser Gly Ser Tyr Leu
Ala Leu Ser Ser20 25 30gtg gtg gcc ctg
gga gcc aac atc atc tgc aac aag att cct ggc ctg 624Val Val Ala Leu
Gly Ala Asn Ile Ile Cys Asn Lys Ile Pro Gly Leu35 40
45gcc cca cgg cag cgt gcc atc tgc cag agc cga ccc gat gcc
atc att 672Ala Pro Arg Gln Arg Ala Ile Cys Gln Ser Arg Pro Asp Ala
Ile Ile50 55 60gtg atc ggg gag ggg gcg
cag atg ggc atc gac gag tgc cag cac cag 720Val Ile Gly Glu Gly Ala
Gln Met Gly Ile Asp Glu Cys Gln His Gln65 70
75 80ttc cga ttc ggc cgc tgg aac tgc tcc gcc ctg
ggc gag aag acc gtc 768Phe Arg Phe Gly Arg Trp Asn Cys Ser Ala Leu
Gly Glu Lys Thr Val85 90 95ttc ggg caa
gaa ctc cga gta ggg agt cga gag gct gcc ttc acc tat 816Phe Gly Gln
Glu Leu Arg Val Gly Ser Arg Glu Ala Ala Phe Thr Tyr100
105 110gcc atc acg gcg gcg ggc gtg gcg cat gct gtc acc
gct gcc tgc agc 864Ala Ile Thr Ala Ala Gly Val Ala His Ala Val Thr
Ala Ala Cys Ser115 120 125cag ggc aat ctg
agc aat tgt ggc tgt gac cgg gag aag caa ggc tac 912Gln Gly Asn Leu
Ser Asn Cys Gly Cys Asp Arg Glu Lys Gln Gly Tyr130 135
140tac aac cag gcg gaa ggc tgg aag tgg ggg ggc tgc tca gcg
gac gtc 960Tyr Asn Gln Ala Glu Gly Trp Lys Trp Gly Gly Cys Ser Ala
Asp Val145 150 155 160cgc
tac ggc atc gac ttt tct cgt cgc ttt gtg gat gcc cgt gag atc 1008Arg
Tyr Gly Ile Asp Phe Ser Arg Arg Phe Val Asp Ala Arg Glu Ile165
170 175aaa aag aac gcc agg cgc ctc atg aac ctt cac
aac aat gag gcg ggc 1056Lys Lys Asn Ala Arg Arg Leu Met Asn Leu His
Asn Asn Glu Ala Gly180 185 190aga aag gtt
ctg gag gac cgc atg aag ctg gaa tgt aag tgt cac ggt 1104Arg Lys Val
Leu Glu Asp Arg Met Lys Leu Glu Cys Lys Cys His Gly195
200 205gtg tca ggc tcc tgt acc acc aaa act tgc tgg acc
acg cta cct aag 1152Val Ser Gly Ser Cys Thr Thr Lys Thr Cys Trp Thr
Thr Leu Pro Lys210 215 220ttc cgc gag gtg
ggc cac ctg ctc aag gag aag tac aac gca gcg gtg 1200Phe Arg Glu Val
Gly His Leu Leu Lys Glu Lys Tyr Asn Ala Ala Val225 230
235 240cag gtg gag gtg gtg cga gcc agc cgc
ctg cgc cag ccc acc ttc ctg 1248Gln Val Glu Val Val Arg Ala Ser Arg
Leu Arg Gln Pro Thr Phe Leu245 250 255cgc
atc aag cag cta cgc agc tac cag aag cct atg gag acg gac ctg 1296Arg
Ile Lys Gln Leu Arg Ser Tyr Gln Lys Pro Met Glu Thr Asp Leu260
265 270gtg tac atc gag aag tcg ccc aac tac tgc gag
gag gac gcg gcc acg 1344Val Tyr Ile Glu Lys Ser Pro Asn Tyr Cys Glu
Glu Asp Ala Ala Thr275 280 285ggc agc gtg
ggc acg cag ggc cgt ctg tgc aac cgc acc tcg ccg ggg 1392Gly Ser Val
Gly Thr Gln Gly Arg Leu Cys Asn Arg Thr Ser Pro Gly290
295 300gcc gac ggc tgt gac acc atg tgc tgc ggc cgc ggc
tac aac acg cac 1440Ala Asp Gly Cys Asp Thr Met Cys Cys Gly Arg Gly
Tyr Asn Thr His305 310 315
320cag tac acc aag gtg tgg cag tgt aac tgc aaa ttc cac tgg tgt tgc
1488Gln Tyr Thr Lys Val Trp Gln Cys Asn Cys Lys Phe His Trp Cys Cys325
330 335ttc gtc aag tgc aac acg tgc agc gag
cgc acc gag gtc ttc acc tgc 1536Phe Val Lys Cys Asn Thr Cys Ser Glu
Arg Thr Glu Val Phe Thr Cys340 345 350aag
tga ggctcccgcg caggcgcgct cggcccctgc cgaccctgcg gccctcgcca 1592Lys
*ttattttgca catccttctt tgcttctgga gctgccagct gcaggcacag gagggtgggg
1652atagaggtgg ggagctcgag atactccagg ctccttccta ctcgctctgt ccccgcccag
1712catccaaggt caacgcaatg gtggtctggt acccaatgga gacaaatccc tttacttctc
1772tttgggaaag tgaaccacaa agggaccatg agactctgag ggtcacctcc ctgcctgtga
1832ctggacacag aaaggccaca cccaccagtc acactcaaaa cggtttcctg ggctgtttcc
1892tgccggccct gggcagtgtg gatggatgtt gacaaaatta tttatgtttt cttagcatca
1952gatgaggact cagtactaac gactgggtag ccagacctaa ccctatttga ggacaccctt
2012ccctcactcc tcccggcccc tccctgcagg gtcctctgct ccttgcagaa ctcgaggatg
2072tcagaattgg cacggaagct ggctggtggg gggactcctt atcagcacct tgggaggggc
2132ttggtggccc tacaaggcct gagatggccg cagaggacag ccaatcttcc attccatttg
2192gagactgtca tgcaaatcaa atgtcccttg tgtcaggctc caggcatgcc tcgtcctctc
2252cctggtcctt caccctccca gcctgctgcc aacctccacc tccagtttac aaattctctt
2312ctcctctgga gccaacctga cacccaggac tgccccacag gttcaggaga ggtcagggac
2372agttgcccca catgacagat ggacagaggg caatctgaag atttactgga gaccccacgg
2432ctctgtgaaa taaatatact gacacagccc catccagccc aactctggaa gttgccaggg
2492tgatgggagg ctgcaccccc ttttcagtaa aaaaaaaaaa aaaaaaaaaa aa
25442353PRTMus musculus 2Met Leu Leu Leu Ser Pro Arg Ser Ala Leu Val Ser
Val Tyr Cys Pro1 5 10
15Gln Ile Phe Leu Leu Leu Ser Ser Gly Ser Tyr Leu Ala Leu Ser Ser20
25 30Val Val Ala Leu Gly Ala Asn Ile Ile Cys
Asn Lys Ile Pro Gly Leu35 40 45Ala Pro
Arg Gln Arg Ala Ile Cys Gln Ser Arg Pro Asp Ala Ile Ile50
55 60Val Ile Gly Glu Gly Ala Gln Met Gly Ile Asp Glu
Cys Gln His Gln65 70 75
80Phe Arg Phe Gly Arg Trp Asn Cys Ser Ala Leu Gly Glu Lys Thr Val85
90 95Phe Gly Gln Glu Leu Arg Val Gly Ser Arg
Glu Ala Ala Phe Thr Tyr100 105 110Ala Ile
Thr Ala Ala Gly Val Ala His Ala Val Thr Ala Ala Cys Ser115
120 125Gln Gly Asn Leu Ser Asn Cys Gly Cys Asp Arg Glu
Lys Gln Gly Tyr130 135 140Tyr Asn Gln Ala
Glu Gly Trp Lys Trp Gly Gly Cys Ser Ala Asp Val145 150
155 160Arg Tyr Gly Ile Asp Phe Ser Arg Arg
Phe Val Asp Ala Arg Glu Ile165 170 175Lys
Lys Asn Ala Arg Arg Leu Met Asn Leu His Asn Asn Glu Ala Gly180
185 190Arg Lys Val Leu Glu Asp Arg Met Lys Leu Glu
Cys Lys Cys His Gly195 200 205Val Ser Gly
Ser Cys Thr Thr Lys Thr Cys Trp Thr Thr Leu Pro Lys210
215 220Phe Arg Glu Val Gly His Leu Leu Lys Glu Lys Tyr
Asn Ala Ala Val225 230 235
240Gln Val Glu Val Val Arg Ala Ser Arg Leu Arg Gln Pro Thr Phe Leu245
250 255Arg Ile Lys Gln Leu Arg Ser Tyr Gln
Lys Pro Met Glu Thr Asp Leu260 265 270Val
Tyr Ile Glu Lys Ser Pro Asn Tyr Cys Glu Glu Asp Ala Ala Thr275
280 285Gly Ser Val Gly Thr Gln Gly Arg Leu Cys Asn
Arg Thr Ser Pro Gly290 295 300Ala Asp Gly
Cys Asp Thr Met Cys Cys Gly Arg Gly Tyr Asn Thr His305
310 315 320Gln Tyr Thr Lys Val Trp Gln
Cys Asn Cys Lys Phe His Trp Cys Cys325 330
335Phe Val Lys Cys Asn Thr Cys Ser Glu Arg Thr Glu Val Phe Thr Cys340
345 350Lys3353PRTMus musculus 3Met Leu Leu
Leu Ser Pro Arg Ser Ala Leu Val Ser Val Tyr Cys Pro1 5
10 15Gln Ile Phe Leu Leu Leu Ser Ser Gly Ser
Tyr Leu Ala Leu Ser Ser20 25 30Val Val
Ala Leu Gly Ala Asn Ile Ile Cys Asn Lys Ile Pro Gly Leu35
40 45Ala Pro Arg Gln Arg Ala Ile Cys Gln Ser Arg Pro
Asp Ala Ile Ile50 55 60Val Ile Gly Glu
Gly Ala Gln Met Gly Ile Asp Glu Cys Gln His Gln65 70
75 80Phe Arg Phe Gly Arg Trp Asn Cys Ser
Ala Leu Gly Glu Lys Thr Val85 90 95Phe
Gly Gln Glu Leu Arg Val Gly Ser Arg Glu Ala Ala Phe Thr Tyr100
105 110Ala Ile Thr Ala Ala Gly Val Ala His Ala Val
Thr Ala Ala Cys Ser115 120 125Gln Gly Asn
Leu Ser Asn Cys Gly Cys Asp Arg Glu Lys Gln Gly Tyr130
135 140Tyr Asn Gln Ala Glu Gly Trp Lys Trp Gly Gly Cys
Ser Ala Asp Val145 150 155
160Arg Tyr Gly Ile Asp Phe Ser Arg Arg Phe Val Asp Ala Arg Glu Ile165
170 175Lys Lys Asn Ala Arg Arg Leu Met Asn
Leu His Asn Asn Glu Ala Gly180 185 190Arg
Lys Val Leu Glu Asp Arg Met Lys Leu Glu Cys Lys Cys His Gly195
200 205Val Ser Gly Ser Cys Thr Thr Lys Thr Cys Trp
Thr Thr Leu Pro Lys210 215 220Phe Arg Glu
Val Gly His Leu Leu Lys Glu Lys Tyr Asn Ala Ala Val225
230 235 240Gln Val Glu Val Val Arg Ala
Ser Arg Leu Arg Gln Pro Thr Phe Leu245 250
255Arg Ile Lys Gln Leu Arg Ser Tyr Gln Lys Pro Met Glu Thr Asp Leu260
265 270Val Tyr Ile Glu Lys Ser Pro Asn Tyr
Cys Glu Glu Asp Ala Ala Thr275 280 285Gly
Ser Val Gly Thr Gln Gly Arg Leu Cys Asn Arg Thr Ser Pro Gly290
295 300Ala Asp Gly Cys Asp Thr Met Cys Cys Gly Arg
Gly Tyr Asn Thr His305 310 315
320Gln Tyr Thr Lys Val Trp Gln Cys Asn Cys Lys Phe His Trp Cys
Cys325 330 335Phe Val Lys Cys Asn Thr Cys
Ser Glu Arg Thr Glu Val Phe Thr Cys340 345
350Lys42160DNAHomo sapiensCDS(6)...(1055)Wnt7b 4ggatc atg cac aga aac
ttt cgc aag tgg att ttc tac gtg ttt ctc tgc 50Met His Arg Asn Phe Arg
Lys Trp Ile Phe Tyr Val Phe Leu Cys1 5 10
15ttt ggc gtc ctg tac gtg aag ctc gga gca ctg tca tcc
gtg gtg gcc 98Phe Gly Val Leu Tyr Val Lys Leu Gly Ala Leu Ser Ser
Val Val Ala20 25 30ctg gga gcc aac atc
atc tgc aac aag att cct ggc cta gcc ccg cgg 146Leu Gly Ala Asn Ile
Ile Cys Asn Lys Ile Pro Gly Leu Ala Pro Arg35 40
45cag cgt gcc atc tgc cag agt cgg ccc gat gcc atc att gtg att
ggg 194Gln Arg Ala Ile Cys Gln Ser Arg Pro Asp Ala Ile Ile Val Ile
Gly50 55 60gag ggg gcg cag atg ggc atc
aac gag tgc cag tac cag ttc cgc ttc 242Glu Gly Ala Gln Met Gly Ile
Asn Glu Cys Gln Tyr Gln Phe Arg Phe65 70
75gga cgc tgg aac tgc tct gcc ctc ggc gag aag acc gtc ttc ggg caa
290Gly Arg Trp Asn Cys Ser Ala Leu Gly Glu Lys Thr Val Phe Gly Gln80
85 90 95gag ctc cga gta ggg
agc cgt gag gct gcc ttc acg tac gcc atc acc 338Glu Leu Arg Val Gly
Ser Arg Glu Ala Ala Phe Thr Tyr Ala Ile Thr100 105
110gcg gct ggc gtg gcg cac gcc gtc acc gct gcc tgc agc caa ggg
aac 386Ala Ala Gly Val Ala His Ala Val Thr Ala Ala Cys Ser Gln Gly
Asn115 120 125ctg agc aac tgc ggc tgc gac
cgc gag aag cag ggc tac tac aac caa 434Leu Ser Asn Cys Gly Cys Asp
Arg Glu Lys Gln Gly Tyr Tyr Asn Gln130 135
140gcc gag ggc tgg aag tgg ggc ggc tgc tcg gcc gac gtg cgt tac ggc
482Ala Glu Gly Trp Lys Trp Gly Gly Cys Ser Ala Asp Val Arg Tyr Gly145
150 155atc gac ttc tcc cgg cgc ttc gtg gac
gct cgg gag atc aag aag aac 530Ile Asp Phe Ser Arg Arg Phe Val Asp
Ala Arg Glu Ile Lys Lys Asn160 165 170
175gcg cgg cgc ctc atg aac ctg cat aac aat gag gcc ggc agg
aag gtt 578Ala Arg Arg Leu Met Asn Leu His Asn Asn Glu Ala Gly Arg
Lys Val180 185 190cta gag gac cgg atg cag
ctg gag tgc aag tgc cac ggc gtg tct ggc 626Leu Glu Asp Arg Met Gln
Leu Glu Cys Lys Cys His Gly Val Ser Gly195 200
205tcc tgc acc acc aaa acc tgc tgg acc acg ctg ccc aag ttc cga gag
674Ser Cys Thr Thr Lys Thr Cys Trp Thr Thr Leu Pro Lys Phe Arg Glu210
215 220gtg ggc cac ctg ctg aag gag aag tac
aac gcg gcc gtg cag gtg gag 722Val Gly His Leu Leu Lys Glu Lys Tyr
Asn Ala Ala Val Gln Val Glu225 230 235gtg
gtg cgg gcc agc cgt ctg cgg cag ccc acc ttc ctg cgc atc aaa 770Val
Val Arg Ala Ser Arg Leu Arg Gln Pro Thr Phe Leu Arg Ile Lys240
245 250 255cag ctg cgc agc tat cag
aag ccc atg gag aca gac ctg gtg tac att 818Gln Leu Arg Ser Tyr Gln
Lys Pro Met Glu Thr Asp Leu Val Tyr Ile260 265
270gag aag tcg ccc aac tac tgc gag gag gac gcg gcc acg ggc agc gtg
866Glu Lys Ser Pro Asn Tyr Cys Glu Glu Asp Ala Ala Thr Gly Ser Val275
280 285ggc acg cag ggc cgt ctc tgc aac cgc
acg tcg ccc ggc gcg gac ggc 914Gly Thr Gln Gly Arg Leu Cys Asn Arg
Thr Ser Pro Gly Ala Asp Gly290 295 300tgt
gac acc atg tgc tgc ggc cga ggc tac aac acc cac cag tac acc 962Cys
Asp Thr Met Cys Cys Gly Arg Gly Tyr Asn Thr His Gln Tyr Thr305
310 315aag gtg tgg cag tgc aac tgc aaa ttc cac tgg
tgc tgc ttc gtc aag 1010Lys Val Trp Gln Cys Asn Cys Lys Phe His Trp
Cys Cys Phe Val Lys320 325 330
335tgc aac acc tgc agc gag cgc acc gag gtc ttc acc tgc aag tga
1055Cys Asn Thr Cys Ser Glu Arg Thr Glu Val Phe Thr Cys Lys *340
345ggccaggccc ggaggcggcc gcgggcaccc tggaacccgg cggcattttg
cacatccact 1115cctcaccttc cctgccttgg tgctgccagc agcagacata gacgggtgca
gaagcgggga 1175gctccaggtg caggagggca ccggccgggg cccacgccct ctgcccgcct
ccctggggct 1235ccttcctgcc acctcctccc atcacctcct gcggcagaac agcacccgtg
acccacccag 1295agagcaaggc caggggtctt ggtgctcccc gacggggccc ggcaagttct
ctttcttctc 1355tctgggaaaa tgaacgtcca ggacacacct gtatcccaga gagcaaagtg
atgaggagac 1415tgagcgtccc cagccccacc tggcggcatg gacacagaaa agctacgccg
gctggcctct 1475ccagaccagt tcccaggctg ggtctgccgc tgggccctgg ggcggtgggg
acagatgttg 1535acacaaatta tttatgtttt cttagtatca gaagaggatt ctcggcacta
acacatagcc 1595agtcctaact ccgtactctg tgtcagccca tcccctagac accctctgtt
tcctttcccg 1655gccccacctg gccggccctc tgcccctgca gagctgaggc agcctggggt
tgatggggac 1715cacgcggtgc ctgcaggtcc tagaagtgag ctgggcaggg gctcttcaga
ccacacagcc 1775ctgaccgggc cttggaggag agccatggac aggctcctcc atgccgtctt
tccttctttt 1835gaaaatccta tcaatggctg ggcgcggtgg ctcacacctg taatcccagc
actttgggag 1895accgaggcag gtggatcacc tgaggtcagg agttcgagac cagcctggcc
aacgtggtga 1955aaccctgtct ctactaaaaa tacaaaaatt agctgggcgt ggtggcgtgc
acctgtaatc 2015ccagctactc aggaggctga gacaggacac ttgcttgaac ccgggaggtg
gaggttgcaa 2075tgagccaaga ttgtgccact gtattccaac ttgggtgaca gagcacgact
ctgtctcaaa 2135aaaaaaaaaa aaaaaaaaaa aaaaa
21605349PRTHomo sapiens 5Met His Arg Asn Phe Arg Lys Trp Ile
Phe Tyr Val Phe Leu Cys Phe1 5 10
15Gly Val Leu Tyr Val Lys Leu Gly Ala Leu Ser Ser Val Val Ala
Leu20 25 30Gly Ala Asn Ile Ile Cys Asn
Lys Ile Pro Gly Leu Ala Pro Arg Gln35 40
45Arg Ala Ile Cys Gln Ser Arg Pro Asp Ala Ile Ile Val Ile Gly Glu50
55 60Gly Ala Gln Met Gly Ile Asn Glu Cys Gln
Tyr Gln Phe Arg Phe Gly65 70 75
80Arg Trp Asn Cys Ser Ala Leu Gly Glu Lys Thr Val Phe Gly Gln
Glu85 90 95Leu Arg Val Gly Ser Arg Glu
Ala Ala Phe Thr Tyr Ala Ile Thr Ala100 105
110Ala Gly Val Ala His Ala Val Thr Ala Ala Cys Ser Gln Gly Asn Leu115
120 125Ser Asn Cys Gly Cys Asp Arg Glu Lys
Gln Gly Tyr Tyr Asn Gln Ala130 135 140Glu
Gly Trp Lys Trp Gly Gly Cys Ser Ala Asp Val Arg Tyr Gly Ile145
150 155 160Asp Phe Ser Arg Arg Phe
Val Asp Ala Arg Glu Ile Lys Lys Asn Ala165 170
175Arg Arg Leu Met Asn Leu His Asn Asn Glu Ala Gly Arg Lys Val
Leu180 185 190Glu Asp Arg Met Gln Leu Glu
Cys Lys Cys His Gly Val Ser Gly Ser195 200
205Cys Thr Thr Lys Thr Cys Trp Thr Thr Leu Pro Lys Phe Arg Glu Val210
215 220Gly His Leu Leu Lys Glu Lys Tyr Asn
Ala Ala Val Gln Val Glu Val225 230 235
240Val Arg Ala Ser Arg Leu Arg Gln Pro Thr Phe Leu Arg Ile
Lys Gln245 250 255Leu Arg Ser Tyr Gln Lys
Pro Met Glu Thr Asp Leu Val Tyr Ile Glu260 265
270Lys Ser Pro Asn Tyr Cys Glu Glu Asp Ala Ala Thr Gly Ser Val
Gly275 280 285Thr Gln Gly Arg Leu Cys Asn
Arg Thr Ser Pro Gly Ala Asp Gly Cys290 295
300Asp Thr Met Cys Cys Gly Arg Gly Tyr Asn Thr His Gln Tyr Thr Lys305
310 315 320Val Trp Gln Cys
Asn Cys Lys Phe His Trp Cys Cys Phe Val Lys Cys325 330
335Asn Thr Cys Ser Glu Arg Thr Glu Val Phe Thr Cys Lys340
3456349PRTHomo sapiens 6Met His Arg Asn Phe Arg Lys Trp Ile
Phe Tyr Val Phe Leu Cys Phe1 5 10
15Gly Val Leu Tyr Val Lys Leu Gly Ala Leu Ser Ser Val Val Ala
Leu20 25 30Gly Ala Asn Ile Ile Cys Asn
Lys Ile Pro Gly Leu Ala Pro Arg Gln35 40
45Arg Ala Ile Cys Gln Ser Arg Pro Asp Ala Ile Ile Val Ile Gly Glu50
55 60Gly Ala Gln Met Gly Ile Asn Glu Cys Gln
Tyr Gln Phe Arg Phe Gly65 70 75
80Arg Trp Asn Cys Ser Ala Leu Gly Glu Lys Thr Val Phe Gly Gln
Glu85 90 95Leu Arg Val Gly Ser Arg Glu
Ala Ala Phe Thr Tyr Ala Ile Thr Ala100 105
110Ala Gly Val Ala His Ala Val Thr Ala Ala Cys Ser Gln Gly Asn Leu115
120 125Ser Asn Cys Gly Cys Asp Arg Glu Lys
Gln Gly Tyr Tyr Asn Gln Ala130 135 140Glu
Gly Trp Lys Trp Gly Gly Cys Ser Ala Asp Val Arg Tyr Gly Ile145
150 155 160Asp Phe Ser Arg Arg Phe
Val Asp Ala Arg Glu Ile Lys Lys Asn Ala165 170
175Arg Arg Leu Met Asn Leu His Asn Asn Glu Ala Gly Arg Lys Val
Leu180 185 190Glu Asp Arg Met Gln Leu Glu
Cys Lys Cys His Gly Val Ser Gly Ser195 200
205Cys Thr Thr Lys Thr Cys Trp Thr Thr Leu Pro Lys Phe Arg Glu Val210
215 220Gly His Leu Leu Lys Glu Lys Tyr Asn
Ala Ala Val Gln Val Glu Val225 230 235
240Val Arg Ala Ser Arg Leu Arg Gln Pro Thr Phe Leu Arg Ile
Lys Gln245 250 255Leu Arg Ser Tyr Gln Lys
Pro Met Glu Thr Asp Leu Val Tyr Ile Glu260 265
270Lys Ser Pro Asn Tyr Cys Glu Glu Asp Ala Ala Thr Gly Ser Val
Gly275 280 285Thr Gln Gly Arg Leu Cys Asn
Arg Thr Ser Pro Gly Ala Asp Gly Cys290 295
300Asp Thr Met Cys Cys Gly Arg Gly Tyr Asn Thr His Gln Tyr Thr Lys305
310 315 320Val Trp Gln Cys
Asn Cys Lys Phe His Trp Cys Cys Phe Val Lys Cys325 330
335Asn Thr Cys Ser Glu Arg Thr Glu Val Phe Thr Cys Lys340
34572475DNAMus musculusCDS(274)...(1764)Angiopoietin 2
7gatactgaca ctgtagactc aggggagaaa caaagagtcc gtgcagacct ctggagtgag
60cagggctgct ccttcctctc aggacagctc cgagtgtgcc ggggagaaga gaagagaaga
120gacaggcact gggaaagagc ctgctgcggg acggagaagg ctctcactga tggacttatt
180cacacggcac agccctgtgc cttagacagc agctgagagc tcaggacgca agtttgctga
240actcacagtt tagaacccaa aaagagagag aga atg tgg cag atc att ttc cta
294Met Trp Gln Ile Ile Phe Leu1 5act ttt ggc tgg gat ctt
gtc ttg gcc tca gcc tac agt aac ttt agg 342Thr Phe Gly Trp Asp Leu
Val Leu Ala Ser Ala Tyr Ser Asn Phe Arg10 15
20aag agc gtg gac agc aca ggc aga agg cag tac cag gtc cag aac gga
390Lys Ser Val Asp Ser Thr Gly Arg Arg Gln Tyr Gln Val Gln Asn Gly25
30 35ccc tgc agc tac acg ttc ctg ctg ccg
gag acc gac agc tgc cga tct 438Pro Cys Ser Tyr Thr Phe Leu Leu Pro
Glu Thr Asp Ser Cys Arg Ser40 45 50
55tcc tcc agc ccc tac atg tcc aat gcc gtg cag agg gat gca
ccc ctc 486Ser Ser Ser Pro Tyr Met Ser Asn Ala Val Gln Arg Asp Ala
Pro Leu60 65 70gac tac gac gac tca gtg
caa agg ctg cag gtg ctg gag aac att cta 534Asp Tyr Asp Asp Ser Val
Gln Arg Leu Gln Val Leu Glu Asn Ile Leu75 80
85gag aac aac aca cag tgg ctg atg aag ctg gag aat tac att cag gac
582Glu Asn Asn Thr Gln Trp Leu Met Lys Leu Glu Asn Tyr Ile Gln Asp90
95 100aac atg aag aag gag atg gtg gag atc
caa cag aat gtg gtg cag aac 630Asn Met Lys Lys Glu Met Val Glu Ile
Gln Gln Asn Val Val Gln Asn105 110 115cag
aca gct gtg atg ata gag att gga acc agc ttg ctg aac cag aca 678Gln
Thr Ala Val Met Ile Glu Ile Gly Thr Ser Leu Leu Asn Gln Thr120
125 130 135gca gca caa act cgg aaa
ctg act gat gtg gaa gcc caa gta cta aac 726Ala Ala Gln Thr Arg Lys
Leu Thr Asp Val Glu Ala Gln Val Leu Asn140 145
150cag acg aca aga ctc gag ctg cag ctt ctc caa cat tct att tct acc
774Gln Thr Thr Arg Leu Glu Leu Gln Leu Leu Gln His Ser Ile Ser Thr155
160 165aac aaa ttg gaa aag cag att ttg gat
cag acc agt gaa ata aac aag 822Asn Lys Leu Glu Lys Gln Ile Leu Asp
Gln Thr Ser Glu Ile Asn Lys170 175 180cta
caa aat aag aac agc ttc cta gaa cag aaa gtt ctg gac atg gag 870Leu
Gln Asn Lys Asn Ser Phe Leu Glu Gln Lys Val Leu Asp Met Glu185
190 195ggc aag cac agc gag cag cta cag tcc atg aag
gag cag aag gac gag 918Gly Lys His Ser Glu Gln Leu Gln Ser Met Lys
Glu Gln Lys Asp Glu200 205 210
215ctc cag gtg ctg gtg tcc aag cag agc tct gtc att gac gag ctg gag
966Leu Gln Val Leu Val Ser Lys Gln Ser Ser Val Ile Asp Glu Leu Glu220
225 230aag aag ctg gtg aca gcc acg gtc aac
aac tcg ctc ctt cag aag cag 1014Lys Lys Leu Val Thr Ala Thr Val Asn
Asn Ser Leu Leu Gln Lys Gln235 240 245cag
cat gac cta atg gag acc gtc aac agc ttg ctg acc atg atg tca 1062Gln
His Asp Leu Met Glu Thr Val Asn Ser Leu Leu Thr Met Met Ser250
255 260tca ccc aac tcc aag agc tcg gtt gct atc cgt
aaa gaa gag caa acc 1110Ser Pro Asn Ser Lys Ser Ser Val Ala Ile Arg
Lys Glu Glu Gln Thr265 270 275acc ttc aga
gac tgt gcg gaa atc ttc aag tca gga ctc acc acc agt 1158Thr Phe Arg
Asp Cys Ala Glu Ile Phe Lys Ser Gly Leu Thr Thr Ser280
285 290 295ggc atc tac aca ctg acc ttc
ccc aac tcc aca gag gag atc aag gcc 1206Gly Ile Tyr Thr Leu Thr Phe
Pro Asn Ser Thr Glu Glu Ile Lys Ala300 305
310tac tgt gac atg gac gtg ggt gga gga ggg tgg aca gtc atc caa cac
1254Tyr Cys Asp Met Asp Val Gly Gly Gly Gly Trp Thr Val Ile Gln His315
320 325cga gaa gat ggc agt gtg gac ttc cag
agg acg tgg aaa gaa tac aaa 1302Arg Glu Asp Gly Ser Val Asp Phe Gln
Arg Thr Trp Lys Glu Tyr Lys330 335 340gag
ggc ttc ggg agc cct ctg gga gag tac tgg ctg ggc aat gag ttt 1350Glu
Gly Phe Gly Ser Pro Leu Gly Glu Tyr Trp Leu Gly Asn Glu Phe345
350 355gtc tcc cag ctg acc ggt cag cac cgc tac gtg
ctt aag atc cag ctg 1398Val Ser Gln Leu Thr Gly Gln His Arg Tyr Val
Leu Lys Ile Gln Leu360 365 370
375aag gac tgg gaa ggc aac gag gcg cat tcg ctg tat gat cac ttc tac
1446Lys Asp Trp Glu Gly Asn Glu Ala His Ser Leu Tyr Asp His Phe Tyr380
385 390ctc gct ggt gaa gag tcc aac tac agg
att cac ctt aca gga ctc acg 1494Leu Ala Gly Glu Glu Ser Asn Tyr Arg
Ile His Leu Thr Gly Leu Thr395 400 405ggg
acc gcg ggc aaa ata agt agc atc agc caa cca gga agt gat ttt 1542Gly
Thr Ala Gly Lys Ile Ser Ser Ile Ser Gln Pro Gly Ser Asp Phe410
415 420agc aca aag gat tcg gac aat gac aaa tgc atc
tgc aag tgt tcc cag 1590Ser Thr Lys Asp Ser Asp Asn Asp Lys Cys Ile
Cys Lys Cys Ser Gln425 430 435atg ctc tca
gga ggc tgg tgg ttt gac gca tgt ggt cct tcc aac ttg 1638Met Leu Ser
Gly Gly Trp Trp Phe Asp Ala Cys Gly Pro Ser Asn Leu440
445 450 455aat gga cag tac tac cca caa
aaa cag aat aca aat aag ttt aac ggt 1686Asn Gly Gln Tyr Tyr Pro Gln
Lys Gln Asn Thr Asn Lys Phe Asn Gly460 465
470atc aag tgg tac tac tgg aag ggg tcc ggc tac tcg ctc aag gcc aca
1734Ile Lys Trp Tyr Tyr Trp Lys Gly Ser Gly Tyr Ser Leu Lys Ala Thr475
480 485acc atg atg atc cgg cca gca gat ttc
taa atgcctgcct acactaccag 1784Thr Met Met Ile Arg Pro Ala Asp Phe
*490 495aagaacttgc tgcatccaaa gattaactcc aaggcactga
gagacaccaa tgcatagcag 1844cccctttcca catcaggaag tgctcctggg ggtggggagg
gtctgtgtgt accagactga 1904agcgcatcac ttaagcctgc accgctaacc aaccaaaggc
actgcagtct ggagaaacac 1964ttctgggaag gttgtggctg aggatcagaa ggacagcgtg
cagactctgt cacagggaag 2024aatgttccgt gggagttcag cagtaaataa ctggaaaaca
gaacacttag atggtgcaga 2084taaatcttgg gaccacattc ctctaagcac ggtttctaga
gtgaatacat tcacagctcg 2144gctgtcacaa tgacaaggcc gtgtcctcgc actgtggcag
ccagtatcca gggatttcta 2204agtggtgggc acaggttatc atctggagaa gcacacattc
attgttttcc tcttgggtgc 2264tttacatgtt catttgaaaa caacacattt acctatcttg
atggcttagt ttttaatggc 2324tggctactat ttactatatg gcaaaaatgc ccacatctct
ggaataacca ccaaataagc 2384gccatgttgg tgaatgcgga gactgtacta ttttgttttc
ttcctggctg ttaaatatga 2444aggtattttt agtaattaaa tataagttat t
24758496PRTMus musculus 8Met Trp Gln Ile Ile Phe
Leu Thr Phe Gly Trp Asp Leu Val Leu Ala1 5
10 15Ser Ala Tyr Ser Asn Phe Arg Lys Ser Val Asp Ser Thr
Gly Arg Arg20 25 30Gln Tyr Gln Val Gln
Asn Gly Pro Cys Ser Tyr Thr Phe Leu Leu Pro35 40
45Glu Thr Asp Ser Cys Arg Ser Ser Ser Ser Pro Tyr Met Ser Asn
Ala50 55 60Val Gln Arg Asp Ala Pro Leu
Asp Tyr Asp Asp Ser Val Gln Arg Leu65 70
75 80Gln Val Leu Glu Asn Ile Leu Glu Asn Asn Thr Gln
Trp Leu Met Lys85 90 95Leu Glu Asn Tyr
Ile Gln Asp Asn Met Lys Lys Glu Met Val Glu Ile100 105
110Gln Gln Asn Val Val Gln Asn Gln Thr Ala Val Met Ile Glu
Ile Gly115 120 125Thr Ser Leu Leu Asn Gln
Thr Ala Ala Gln Thr Arg Lys Leu Thr Asp130 135
140Val Glu Ala Gln Val Leu Asn Gln Thr Thr Arg Leu Glu Leu Gln
Leu145 150 155 160Leu Gln
His Ser Ile Ser Thr Asn Lys Leu Glu Lys Gln Ile Leu Asp165
170 175Gln Thr Ser Glu Ile Asn Lys Leu Gln Asn Lys Asn
Ser Phe Leu Glu180 185 190Gln Lys Val Leu
Asp Met Glu Gly Lys His Ser Glu Gln Leu Gln Ser195 200
205Met Lys Glu Gln Lys Asp Glu Leu Gln Val Leu Val Ser Lys
Gln Ser210 215 220Ser Val Ile Asp Glu Leu
Glu Lys Lys Leu Val Thr Ala Thr Val Asn225 230
235 240Asn Ser Leu Leu Gln Lys Gln Gln His Asp Leu
Met Glu Thr Val Asn245 250 255Ser Leu Leu
Thr Met Met Ser Ser Pro Asn Ser Lys Ser Ser Val Ala260
265 270Ile Arg Lys Glu Glu Gln Thr Thr Phe Arg Asp Cys
Ala Glu Ile Phe275 280 285Lys Ser Gly Leu
Thr Thr Ser Gly Ile Tyr Thr Leu Thr Phe Pro Asn290 295
300Ser Thr Glu Glu Ile Lys Ala Tyr Cys Asp Met Asp Val Gly
Gly Gly305 310 315 320Gly
Trp Thr Val Ile Gln His Arg Glu Asp Gly Ser Val Asp Phe Gln325
330 335Arg Thr Trp Lys Glu Tyr Lys Glu Gly Phe Gly
Ser Pro Leu Gly Glu340 345 350Tyr Trp Leu
Gly Asn Glu Phe Val Ser Gln Leu Thr Gly Gln His Arg355
360 365Tyr Val Leu Lys Ile Gln Leu Lys Asp Trp Glu Gly
Asn Glu Ala His370 375 380Ser Leu Tyr Asp
His Phe Tyr Leu Ala Gly Glu Glu Ser Asn Tyr Arg385 390
395 400Ile His Leu Thr Gly Leu Thr Gly Thr
Ala Gly Lys Ile Ser Ser Ile405 410 415Ser
Gln Pro Gly Ser Asp Phe Ser Thr Lys Asp Ser Asp Asn Asp Lys420
425 430Cys Ile Cys Lys Cys Ser Gln Met Leu Ser Gly
Gly Trp Trp Phe Asp435 440 445Ala Cys Gly
Pro Ser Asn Leu Asn Gly Gln Tyr Tyr Pro Gln Lys Gln450
455 460Asn Thr Asn Lys Phe Asn Gly Ile Lys Trp Tyr Tyr
Trp Lys Gly Ser465 470 475
480Gly Tyr Ser Leu Lys Ala Thr Thr Met Met Ile Arg Pro Ala Asp Phe485
490 4959496PRTMus musculus 9Met Trp Gln Ile
Ile Phe Leu Thr Phe Gly Trp Asp Leu Val Leu Ala1 5
10 15Ser Ala Tyr Ser Asn Phe Arg Lys Ser Val Asp
Ser Thr Gly Arg Arg20 25 30Gln Tyr Gln
Val Gln Asn Gly Pro Cys Ser Tyr Thr Phe Leu Leu Pro35 40
45Glu Thr Asp Ser Cys Arg Ser Ser Ser Ser Pro Tyr Met
Ser Asn Ala50 55 60Val Gln Arg Asp Ala
Pro Leu Asp Tyr Asp Asp Ser Val Gln Arg Leu65 70
75 80Gln Val Leu Glu Asn Ile Leu Glu Asn Asn
Thr Gln Trp Leu Met Lys85 90 95Leu Glu
Asn Tyr Ile Gln Asp Asn Met Lys Lys Glu Met Val Glu Ile100
105 110Gln Gln Asn Val Val Gln Asn Gln Thr Ala Val Met
Ile Glu Ile Gly115 120 125Thr Ser Leu Leu
Asn Gln Thr Ala Ala Gln Thr Arg Lys Leu Thr Asp130 135
140Val Glu Ala Gln Val Leu Asn Gln Thr Thr Arg Leu Glu Leu
Gln Leu145 150 155 160Leu
Gln His Ser Ile Ser Thr Asn Lys Leu Glu Lys Gln Ile Leu Asp165
170 175Gln Thr Ser Glu Ile Asn Lys Leu Gln Asn Lys
Asn Ser Phe Leu Glu180 185 190Gln Lys Val
Leu Asp Met Glu Gly Lys His Ser Glu Gln Leu Gln Ser195
200 205Met Lys Glu Gln Lys Asp Glu Leu Gln Val Leu Val
Ser Lys Gln Ser210 215 220Ser Val Ile Asp
Glu Leu Glu Lys Lys Leu Val Thr Ala Thr Val Asn225 230
235 240Asn Ser Leu Leu Gln Lys Gln Gln His
Asp Leu Met Glu Thr Val Asn245 250 255Ser
Leu Leu Thr Met Met Ser Ser Pro Asn Ser Lys Ser Ser Val Ala260
265 270Ile Arg Lys Glu Glu Gln Thr Thr Phe Arg Asp
Cys Ala Glu Ile Phe275 280 285Lys Ser Gly
Leu Thr Thr Ser Gly Ile Tyr Thr Leu Thr Phe Pro Asn290
295 300Ser Thr Glu Glu Ile Lys Ala Tyr Cys Asp Met Asp
Val Gly Gly Gly305 310 315
320Gly Trp Thr Val Ile Gln His Arg Glu Asp Gly Ser Val Asp Phe Gln325
330 335Arg Thr Trp Lys Glu Tyr Lys Glu Gly
Phe Gly Ser Pro Leu Gly Glu340 345 350Tyr
Trp Leu Gly Asn Glu Phe Val Ser Gln Leu Thr Gly Gln His Arg355
360 365Tyr Val Leu Lys Ile Gln Leu Lys Asp Trp Glu
Gly Asn Glu Ala His370 375 380Ser Leu Tyr
Asp His Phe Tyr Leu Ala Gly Glu Glu Ser Asn Tyr Arg385
390 395 400Ile His Leu Thr Gly Leu Thr
Gly Thr Ala Gly Lys Ile Ser Ser Ile405 410
415Ser Gln Pro Gly Ser Asp Phe Ser Thr Lys Asp Ser Asp Asn Asp Lys420
425 430Cys Ile Cys Lys Cys Ser Gln Met Leu
Ser Gly Gly Trp Trp Phe Asp435 440 445Ala
Cys Gly Pro Ser Asn Leu Asn Gly Gln Tyr Tyr Pro Gln Lys Gln450
455 460Asn Thr Asn Lys Phe Asn Gly Ile Lys Trp Tyr
Tyr Trp Lys Gly Ser465 470 475
480Gly Tyr Ser Leu Lys Ala Thr Thr Met Met Ile Arg Pro Ala Asp
Phe485 490 495102269DNAHomo
sapiensCDS(350)...(1840)Angiopoietin 2 10tgggttggtg tttatctcct cccagccttg
agggagggaa caacactgta ggatctgggg 60agagaggaac aaaggaccgt gaaagctgct
ctgtaaaagc tgacacagcc ctcccaagtg 120agcaggactg ttcttcccac tgcaatctga
cagtttactg catgcctgga gagaacacag 180cagtaaaaac caggtttgct actggaaaaa
gaggaaagag aagactttca ttgacggacc 240cagccatggc agcgtagcag ccctgcgttt
cagacggcag cagctcggga ctctggacgt 300gtgtttgccc tcaagtttgc taagctgctg
gtttattact gaagaaaga atg tgg cag 358Met Trp Gln1att gtt ttc ttt act
ctg agc tgt gat ctt gtc ttg gcc gca gcc tat 406Ile Val Phe Phe Thr
Leu Ser Cys Asp Leu Val Leu Ala Ala Ala Tyr5 10
15aac aac ttt cgg aag agc atg gac agc ata gga aag aag caa tat
cag 454Asn Asn Phe Arg Lys Ser Met Asp Ser Ile Gly Lys Lys Gln Tyr
Gln20 25 30 35gtc cag
cat ggg tcc tgc agc tac act ttc ctc ctg cca gag atg gac 502Val Gln
His Gly Ser Cys Ser Tyr Thr Phe Leu Leu Pro Glu Met Asp40
45 50aac tgc cgc tct tcc tcc agc ccc tac gtg tcc aat
gct gtg cag agg 550Asn Cys Arg Ser Ser Ser Ser Pro Tyr Val Ser Asn
Ala Val Gln Arg55 60 65gac gcg ccg ctc
gaa tac gat gac tcg gtg cag agg ctg caa gtg ctg 598Asp Ala Pro Leu
Glu Tyr Asp Asp Ser Val Gln Arg Leu Gln Val Leu70 75
80gag aac atc atg gaa aac aac act cag tgg cta atg aag ctt
gag aat 646Glu Asn Ile Met Glu Asn Asn Thr Gln Trp Leu Met Lys Leu
Glu Asn85 90 95tat atc cag gac aac atg
aag aaa gaa atg gta gag ata cag cag aat 694Tyr Ile Gln Asp Asn Met
Lys Lys Glu Met Val Glu Ile Gln Gln Asn100 105
110 115gca gta cag aac cag acg gct gtg atg ata gaa
ata ggg aca aac ctg 742Ala Val Gln Asn Gln Thr Ala Val Met Ile Glu
Ile Gly Thr Asn Leu120 125 130ttg aac caa
aca gct gag caa acg cgg aag tta act gat gtg gaa gcc 790Leu Asn Gln
Thr Ala Glu Gln Thr Arg Lys Leu Thr Asp Val Glu Ala135
140 145caa gta tta aat cag acc acg aga ctt gaa ctt cag
ctc ttg gaa cac 838Gln Val Leu Asn Gln Thr Thr Arg Leu Glu Leu Gln
Leu Leu Glu His150 155 160tcc ctc tcg aca
aac aaa ttg gaa aaa cag att ttg gac cag acc agt 886Ser Leu Ser Thr
Asn Lys Leu Glu Lys Gln Ile Leu Asp Gln Thr Ser165 170
175gaa ata aac aaa ttg caa gat aag aac agt ttc cta gaa aag
aag gtg 934Glu Ile Asn Lys Leu Gln Asp Lys Asn Ser Phe Leu Glu Lys
Lys Val180 185 190 195cta
gct atg gaa gac aag cac atc atc caa cta cag tca ata aaa gaa 982Leu
Ala Met Glu Asp Lys His Ile Ile Gln Leu Gln Ser Ile Lys Glu200
205 210gag aaa gat cag cta cag gtg tta gta tcc aag
caa aat tcc atc att 1030Glu Lys Asp Gln Leu Gln Val Leu Val Ser Lys
Gln Asn Ser Ile Ile215 220 225gaa gaa cta
gaa aaa aaa ata gtg act gcc acg gtg aat aat tca gtt 1078Glu Glu Leu
Glu Lys Lys Ile Val Thr Ala Thr Val Asn Asn Ser Val230
235 240ctt caa aag cag caa cat gat ctc atg gag aca gtt
aat aac tta ctg 1126Leu Gln Lys Gln Gln His Asp Leu Met Glu Thr Val
Asn Asn Leu Leu245 250 255act atg atg tcc
aca tca aac tca gct aag gac ccc act gtt gct aaa 1174Thr Met Met Ser
Thr Ser Asn Ser Ala Lys Asp Pro Thr Val Ala Lys260 265
270 275gaa gaa caa atc agc ttc aga gac tgt
gct gaa gta ttc aaa tca gga 1222Glu Glu Gln Ile Ser Phe Arg Asp Cys
Ala Glu Val Phe Lys Ser Gly280 285 290cac
acc aca aat ggc atc tac acg tta aca ttc cct aat tct aca gaa 1270His
Thr Thr Asn Gly Ile Tyr Thr Leu Thr Phe Pro Asn Ser Thr Glu295
300 305gag atc aag gcc tac tgt gac atg gaa gct gga
gga ggc ggg tgg aca 1318Glu Ile Lys Ala Tyr Cys Asp Met Glu Ala Gly
Gly Gly Gly Trp Thr310 315 320att att cag
cga cgt gag gat ggc agc gtt gat ttt cag agg act tgg 1366Ile Ile Gln
Arg Arg Glu Asp Gly Ser Val Asp Phe Gln Arg Thr Trp325
330 335aaa gaa tat aaa gtg gga ttt ggt aac cct tca gga
gaa tat tgg ctg 1414Lys Glu Tyr Lys Val Gly Phe Gly Asn Pro Ser Gly
Glu Tyr Trp Leu340 345 350
355gga aat gag ttt gtt tcg caa ctg act aat cag caa cgc tat gtg ctt
1462Gly Asn Glu Phe Val Ser Gln Leu Thr Asn Gln Gln Arg Tyr Val Leu360
365 370aaa ata cac ctt aaa gac tgg gaa ggg
aat gag gct tac tca ttg tat 1510Lys Ile His Leu Lys Asp Trp Glu Gly
Asn Glu Ala Tyr Ser Leu Tyr375 380 385gaa
cat ttc tat ctc tca agt gaa gaa ctc aat tat agg att cac ctt 1558Glu
His Phe Tyr Leu Ser Ser Glu Glu Leu Asn Tyr Arg Ile His Leu390
395 400aaa gga ctt aca ggg aca gcc ggc aaa ata agc
agc atc agc caa cca 1606Lys Gly Leu Thr Gly Thr Ala Gly Lys Ile Ser
Ser Ile Ser Gln Pro405 410 415gga aat gat
ttt agc aca aag gat gga gac aac gac aaa tgt att tgc 1654Gly Asn Asp
Phe Ser Thr Lys Asp Gly Asp Asn Asp Lys Cys Ile Cys420
425 430 435aaa tgt tca caa atg cta aca
gga ggc tgg tgg ttt gat gca tgt ggt 1702Lys Cys Ser Gln Met Leu Thr
Gly Gly Trp Trp Phe Asp Ala Cys Gly440 445
450cct tcc aac ttg aac gga atg tac tat cca cag agg cag aac aca aat
1750Pro Ser Asn Leu Asn Gly Met Tyr Tyr Pro Gln Arg Gln Asn Thr Asn455
460 465aag ttc aac ggc att aaa tgg tac tac
tgg aaa ggc tca ggc tat tcg 1798Lys Phe Asn Gly Ile Lys Trp Tyr Tyr
Trp Lys Gly Ser Gly Tyr Ser470 475 480ctc
aag gcc aca acc atg atg atc cga cca gca gat ttc taa 1840Leu
Lys Ala Thr Thr Met Met Ile Arg Pro Ala Asp Phe *485 490
495acatcccagt ccacctgagg aactgtctcg aactattttc aaagacttaa
gcccagtgca 1900ctgaaagtca cggctgcgca ctgtgtcctc ttccaccaca gagggcgtgt
gctcggtgct 1960gacgggaccc acatgctcca gattagagcc tgtaaacttt atcacttaaa
cttgcatcac 2020ttaacggacc aaagcaagac cctaaacatc cataattgtg attagacaga
acacctatgc 2080aaagatgaac ccgaggctga gaatcagact gacagtttac agacgctgct
gtcacaacca 2140agaatgttat gtgcaagttt atcagtaaat aactggaaaa cagaacactt
atgttataca 2200atacagatca tcttggaact gcattcttct gagcactgtt tatacactgt
gtaaataccc 2260atatgtcct
226911496PRTHomo sapiens 11Met Trp Gln Ile Val Phe Phe Thr Leu
Ser Cys Asp Leu Val Leu Ala1 5 10
15Ala Ala Tyr Asn Asn Phe Arg Lys Ser Met Asp Ser Ile Gly Lys
Lys20 25 30Gln Tyr Gln Val Gln His Gly
Ser Cys Ser Tyr Thr Phe Leu Leu Pro35 40
45Glu Met Asp Asn Cys Arg Ser Ser Ser Ser Pro Tyr Val Ser Asn Ala50
55 60Val Gln Arg Asp Ala Pro Leu Glu Tyr Asp
Asp Ser Val Gln Arg Leu65 70 75
80Gln Val Leu Glu Asn Ile Met Glu Asn Asn Thr Gln Trp Leu Met
Lys85 90 95Leu Glu Asn Tyr Ile Gln Asp
Asn Met Lys Lys Glu Met Val Glu Ile100 105
110Gln Gln Asn Ala Val Gln Asn Gln Thr Ala Val Met Ile Glu Ile Gly115
120 125Thr Asn Leu Leu Asn Gln Thr Ala Glu
Gln Thr Arg Lys Leu Thr Asp130 135 140Val
Glu Ala Gln Val Leu Asn Gln Thr Thr Arg Leu Glu Leu Gln Leu145
150 155 160Leu Glu His Ser Leu Ser
Thr Asn Lys Leu Glu Lys Gln Ile Leu Asp165 170
175Gln Thr Ser Glu Ile Asn Lys Leu Gln Asp Lys Asn Ser Phe Leu
Glu180 185 190Lys Lys Val Leu Ala Met Glu
Asp Lys His Ile Ile Gln Leu Gln Ser195 200
205Ile Lys Glu Glu Lys Asp Gln Leu Gln Val Leu Val Ser Lys Gln Asn210
215 220Ser Ile Ile Glu Glu Leu Glu Lys Lys
Ile Val Thr Ala Thr Val Asn225 230 235
240Asn Ser Val Leu Gln Lys Gln Gln His Asp Leu Met Glu Thr
Val Asn245 250 255Asn Leu Leu Thr Met Met
Ser Thr Ser Asn Ser Ala Lys Asp Pro Thr260 265
270Val Ala Lys Glu Glu Gln Ile Ser Phe Arg Asp Cys Ala Glu Val
Phe275 280 285Lys Ser Gly His Thr Thr Asn
Gly Ile Tyr Thr Leu Thr Phe Pro Asn290 295
300Ser Thr Glu Glu Ile Lys Ala Tyr Cys Asp Met Glu Ala Gly Gly Gly305
310 315 320Gly Trp Thr Ile
Ile Gln Arg Arg Glu Asp Gly Ser Val Asp Phe Gln325 330
335Arg Thr Trp Lys Glu Tyr Lys Val Gly Phe Gly Asn Pro Ser
Gly Glu340 345 350Tyr Trp Leu Gly Asn Glu
Phe Val Ser Gln Leu Thr Asn Gln Gln Arg355 360
365Tyr Val Leu Lys Ile His Leu Lys Asp Trp Glu Gly Asn Glu Ala
Tyr370 375 380Ser Leu Tyr Glu His Phe Tyr
Leu Ser Ser Glu Glu Leu Asn Tyr Arg385 390
395 400Ile His Leu Lys Gly Leu Thr Gly Thr Ala Gly Lys
Ile Ser Ser Ile405 410 415Ser Gln Pro Gly
Asn Asp Phe Ser Thr Lys Asp Gly Asp Asn Asp Lys420 425
430Cys Ile Cys Lys Cys Ser Gln Met Leu Thr Gly Gly Trp Trp
Phe Asp435 440 445Ala Cys Gly Pro Ser Asn
Leu Asn Gly Met Tyr Tyr Pro Gln Arg Gln450 455
460Asn Thr Asn Lys Phe Asn Gly Ile Lys Trp Tyr Tyr Trp Lys Gly
Ser465 470 475 480Gly Tyr
Ser Leu Lys Ala Thr Thr Met Met Ile Arg Pro Ala Asp Phe485
490 49512496PRTHomo sapiens 12Met Trp Gln Ile Val Phe
Phe Thr Leu Ser Cys Asp Leu Val Leu Ala1 5
10 15Ala Ala Tyr Asn Asn Phe Arg Lys Ser Met Asp Ser Ile
Gly Lys Lys20 25 30Gln Tyr Gln Val Gln
His Gly Ser Cys Ser Tyr Thr Phe Leu Leu Pro35 40
45Glu Met Asp Asn Cys Arg Ser Ser Ser Ser Pro Tyr Val Ser Asn
Ala50 55 60Val Gln Arg Asp Ala Pro Leu
Glu Tyr Asp Asp Ser Val Gln Arg Leu65 70
75 80Gln Val Leu Glu Asn Ile Met Glu Asn Asn Thr Gln
Trp Leu Met Lys85 90 95Leu Glu Asn Tyr
Ile Gln Asp Asn Met Lys Lys Glu Met Val Glu Ile100 105
110Gln Gln Asn Ala Val Gln Asn Gln Thr Ala Val Met Ile Glu
Ile Gly115 120 125Thr Asn Leu Leu Asn Gln
Thr Ala Glu Gln Thr Arg Lys Leu Thr Asp130 135
140Val Glu Ala Gln Val Leu Asn Gln Thr Thr Arg Leu Glu Leu Gln
Leu145 150 155 160Leu Glu
His Ser Leu Ser Thr Asn Lys Leu Glu Lys Gln Ile Leu Asp165
170 175Gln Thr Ser Glu Ile Asn Lys Leu Gln Asp Lys Asn
Ser Phe Leu Glu180 185 190Lys Lys Val Leu
Ala Met Glu Asp Lys His Ile Ile Gln Leu Gln Ser195 200
205Ile Lys Glu Glu Lys Asp Gln Leu Gln Val Leu Val Ser Lys
Gln Asn210 215 220Ser Ile Ile Glu Glu Leu
Glu Lys Lys Ile Val Thr Ala Thr Val Asn225 230
235 240Asn Ser Val Leu Gln Lys Gln Gln His Asp Leu
Met Glu Thr Val Asn245 250 255Asn Leu Leu
Thr Met Met Ser Thr Ser Asn Ser Ala Lys Asp Pro Thr260
265 270Val Ala Lys Glu Glu Gln Ile Ser Phe Arg Asp Cys
Ala Glu Val Phe275 280 285Lys Ser Gly His
Thr Thr Asn Gly Ile Tyr Thr Leu Thr Phe Pro Asn290 295
300Ser Thr Glu Glu Ile Lys Ala Tyr Cys Asp Met Glu Ala Gly
Gly Gly305 310 315 320Gly
Trp Thr Ile Ile Gln Arg Arg Glu Asp Gly Ser Val Asp Phe Gln325
330 335Arg Thr Trp Lys Glu Tyr Lys Val Gly Phe Gly
Asn Pro Ser Gly Glu340 345 350Tyr Trp Leu
Gly Asn Glu Phe Val Ser Gln Leu Thr Asn Gln Gln Arg355
360 365Tyr Val Leu Lys Ile His Leu Lys Asp Trp Glu Gly
Asn Glu Ala Tyr370 375 380Ser Leu Tyr Glu
His Phe Tyr Leu Ser Ser Glu Glu Leu Asn Tyr Arg385 390
395 400Ile His Leu Lys Gly Leu Thr Gly Thr
Ala Gly Lys Ile Ser Ser Ile405 410 415Ser
Gln Pro Gly Asn Asp Phe Ser Thr Lys Asp Gly Asp Asn Asp Lys420
425 430Cys Ile Cys Lys Cys Ser Gln Met Leu Thr Gly
Gly Trp Trp Phe Asp435 440 445Ala Cys Gly
Pro Ser Asn Leu Asn Gly Met Tyr Tyr Pro Gln Arg Gln450
455 460Asn Thr Asn Lys Phe Asn Gly Ile Lys Trp Tyr Tyr
Trp Lys Gly Ser465 470 475
480Gly Tyr Ser Leu Lys Ala Thr Thr Met Met Ile Arg Pro Ala Asp Phe485
490 495
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